Pig ileitis subunit vaccine and application thereof

Abstract

The invention discloses a swine ileitis subunit vaccine and application thereof. The subunit vaccine comprises an Omp2 protein as shown in SEQ ID No. 2, an hlyA protein as shown in SEQ ID No. 4, a flgJ protein as shown in SEQ ID No. 6, and a ndK protein as shown in SEQ ID No. 8. The invention further discloses a preparation method of the subunit vaccine. The subunit vaccine comprises the antigen protein of the lawsonia intracellularis (Lawsonia intracellularis) and an adjuvant, and the subunit vaccine comprises the antigen protein of the lawsonia intracellularis (Lawsonia intracellularis) and the adjuvant. Research finds that the prepared subunit vaccine can rapidly induce piglets to generate high-level serum antibodies and can provide a good immune protection effect on infection of Lawsonia intracellularis. Soluble expression of the selected proteins Omp2, hlyA, flgJ and ndK is successfully achieved, mass expression and purification are easy, the production cost is reduced, and the vaccine is an ideal subunit candidate vaccine for porcine ileitis and has good application and development prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, especially the field of veterinary vaccine technology, and specifically relates to the development and application of a porcine ileitis subunit vaccine. Background Technology

[0002] Porcine ileitis, also known as porcine proliferative enteropathy (PPE), is an intestinal infectious disease caused by Lawsonia intracellularis (L. intracellularis, LI). First reported in 1931, it has become widespread in pig farms worldwide. The disease primarily affects growing-finishing pigs aged 6-20 weeks, manifesting mainly as diarrhea, acute hemorrhagic diarrhea, decreased appetite, growth retardation, and reduced feed conversion ratio, causing severe economic losses to the global pig industry. In recent years, the incidence of this disease in intensive pig farms has been steadily increasing, with subclinical infections being common, and some farms reporting PPE positivity rates as high as 98%.

[0003] Currently, clinical prevention and control of PPE mainly relies on drug treatment and vaccination. Adding antibiotics to the feed can control the progression of ileitis when infected pigs show early clinical symptoms. However, with the increasing necessity of reducing, limiting, and even banning antibiotics, vaccination is gradually becoming the primary and effective prevention and control method. Currently, PPE vaccine research is mainly divided into three categories: whole-cell inactivated vaccines, live attenuated vaccines, and subunit vaccines.

[0004] Currently, the commercially available vaccines for swine ileitis include a live attenuated vaccine developed by Boehringer Ingelheim. Ileitis and the inactivated vaccine developed by Merck in the United States ( Ileitis. Live attenuated vaccines have been shown to induce humoral and cellular immune responses in animals, increasing their weight, but they do not provide complete protection. Infected pigs remain carriers for a long time, excreting bacteria-laden feces, increasing the risk of secondary infection. Furthermore, because live attenuated vaccines are active, they cannot be used concurrently with antibiotics. Inactivated vaccines have a short duration of immunity, require multiple vaccinations, and cannot distinguish between vaccine-induced and natural infection. Both live attenuated and inactivated vaccines cannot differentiate between vaccine-induced and natural infection, and their production is difficult and costly due to the demanding culture conditions required for Ileitis. Given the inherent limitations of traditional vaccines, subunit vaccines may be a more promising alternative. Subunit vaccines have clearly defined components, high safety, few side effects, low production costs, and do not exhibit shedding of the virus, making them distinguishable from wild-type infections. Compared to whole-cell vaccines, subunit vaccines target a small number of selected antigens, typically six or fewer. Therefore, a major challenge in developing protein subunit vaccines is determining the optimal antigens for such vaccines.

[0005] Currently, there are reports of recombinant Omp2 subunit vaccines against LI, which are co-expressed in Escherichia coli and have good immunogenicity in mice. Both IgG and IFN-γ levels are elevated, and no PPE lesions such as thickening of the intestinal mucosa or crypt hyperplasia are observed, indicating good immunoprotective efficacy.

[0006] However, bacterial subunit vaccines with a single antigen have limited protective rates. Therefore, it is necessary to provide a commercially available porcine ileitis subunit vaccine that targets different antigens of Lawsonia intracellularis and significantly improves vaccine protection rates. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings and deficiencies of existing vaccine technologies and to provide an effective subunit vaccine against PPE, its preparation method, and its application.

[0008] To achieve the above objectives, the present invention employs the following technical means:

[0009] The first objective of this invention is to provide an intracellular Lawsonia antigen protein, wherein the antigen protein is selected from hlyA protein, flgJ protein, and ndK protein; the amino acid sequence of hlyA protein is shown in SEQ ID No. 4; the amino acid sequence of flgJ protein is shown in SEQ ID No. 6; and the amino acid sequence of ndK protein is shown in SEQ ID No. 8.

[0010] A second object of the present invention is to provide genes encoding the aforementioned intracellular Lawsonia antigen proteins, wherein the nucleotide sequence of the gene encoding the hlyA protein is shown in SEQ ID No. 3, the nucleotide sequence of the gene encoding the flgJ protein is shown in SEQ ID No. 5, and the nucleotide sequence of the gene encoding the ndK protein is shown in SEQ ID No. 7.

[0011] The third objective of this invention is to provide a method for preparing the aforementioned intracellular Lawsonia antigen proteins, wherein the hlyA protein, flgJ protein, and ndK protein are all obtained through prokaryotic expression.

[0012] Furthermore, this includes the following steps:

[0013] (1) Construction of recombinant plasmids: The hlyA, flgJ and ndK genes were inserted between the BamHI and Xho I restriction sites of the prokaryotic expression vector pET-28a, respectively, and transformed into DH5α competent cells. Positive clones were screened, plasmids of positive clones were extracted and sequenced, and plasmids with the correct target fragments were selected and transformed into BL21(DE3) competent cells.

[0014] (2) Recombinant protein expression and purification: positive clones are cultured, protein expression is induced, and the protein is purified by affinity chromatography.

[0015] A fourth objective of this invention is to provide a porcine ileitis subunit vaccine, wherein the porcine ileitis subunit vaccine uses one or more of the aforementioned hlyA protein, flgJ protein, and ndK protein as antigens, or uses a combination of one or more of the hlyA protein, flgJ protein, and ndK protein with Omp2 protein as antigens.

[0016] In a particular embodiment, the vaccine protein includes Omp2, hlyA, flgJ, ndK, Omp2+hlyA combination, Omp2+flgJ combination, and Omp2+ndK combination.

[0017] Furthermore, the amino acid sequence of the Omp2 protein is shown in SEQ ID No. 2, and the nucleotide sequence of the gene encoding the Omp2 protein is shown in SEQ ID No. 1.

[0018] Furthermore, the porcine ileitis subunit vaccine also includes an adjuvant, which is a water-in-oil-in-water adjuvant, preferably ISA201VG.

[0019] Furthermore, the volume ratio of antigen to adjuvant is 1:1.

[0020] The fifth objective of this invention is to provide the application of the aforementioned intracellular Lawsonia antigen protein in the preparation of a subunit vaccine for the prevention of porcine ileitis.

[0021] A sixth objective of this invention is to provide the application of the aforementioned gene encoding intracellular Lawsonia antigen protein in the preparation of a subunit vaccine for the prevention of porcine ileitis.

[0022] The preferred route of administration for the subunit vaccine is intramuscular injection into the back of the neck.

[0023] Preferably, the subunit vaccination regimen of this invention consists of an initial immunization and a booster immunization. The initial immunization is administered at 3 weeks of age, followed by booster immunizations on day 21. The dose of the booster immunization is the same as that of the initial immunization.

[0024] The beneficial effects of this invention are:

[0025] 1. This invention provides a highly efficient, safe, and immune-inducible subunit vaccine. By expressing the full-length key immunogenic proteins Omp2, hlyA, flgJ, and ndK of Lawsonia intracellularis, soluble proteins are obtained. The optimal adjuvant is selected, and immunoprotective efficacy is evaluated in pigs. The protein with the best immunoprotective effect is screened, and a subunit vaccine capable of effectively preventing Lawsonia intracellularis infection is prepared, providing a new technical means for the prevention and control of porcine ileitis.

[0026] The applicant identified four key immunogenic proteins of Lawsonia intracellularis:

[0027] Omp2 protein is an outer membrane protein.

[0028] hlyA protein is a virulence factor with dual functions as a hemolytic and ribosomal RNA methyltransferase.

[0029] flgJ protein is a protein involved in the biosynthesis of flagella in many Gram-negative bacterial pathogens.

[0030] ndK protein is a nucleoside diphosphate kinase.

[0031] 2. This invention uses E. coli prokaryotic expression of intracellular Lawsonia antigen proteins Omp2, hlyA, flgJ, and ndK. After optimization, all of them were successfully expressed in a soluble manner, which is easy to express and purify in large quantities and reduces production costs.

[0032] 3. Animal experiments showed that all immunization groups significantly increased the level of specific IgG antibodies in piglet serum, increased the average daily weight gain, and reduced the bacterial load in feces and ileum tissue. Among them, the combination of Omp2+hlyA and Omp2+ndK provided the best immune protection.

[0033] 4. Subunit vaccines using recombinant proteins of Omp2, hlyA, flgJ, and ndK as vaccine antigens have shown good efficacy in terms of safety and protection. Attached Figure Description

[0034] Figure 1 The images show the PCR identification of recombinant plasmids pGex-6p-1-Omp2, pET-28a-hlyA, pET-28a-flgJ, and pET-28a-ndK.

[0035] Figure A: Lanes: M: DL2000 DNA Marker; 2: Omp2 (891bp); 4: hlyA (753bp); 6: flgJ (681bp);

[0036] Figure B: Lanes: M: DL5000 DNA Marker; 7: ndK (417bp).

[0037] Figure 2 The image shows the SDS-PAGE analysis of the purified recombinant proteins Omp2, hlyA, and flgJ, where: M: protein marker; 1: Omp2 (59 kDa); 2: hlyA (28 kDa); 3: flgJ (26 kDa); 4: ndK (14 kDa).

[0038] Figure 3Figure 1 shows the serum IgG antibody levels in piglets at 0, 21, and 35 days post-immunization and 21 days post-challenge.

[0039] Figure 4 The graph shows the bacterial count in the feces of piglets in each group at 1, 2, and 3 weeks after viral challenge.

[0040] Figure 5 The images show H&E staining and IHC staining of the blank control group, the challenge control group, and the vaccine-immunized group after viral challenge. Detailed Implementation

[0041] Example 1: Construction of protein expression strain

[0042] In this invention, Omp2 (as shown in SEQ ID No. 1, encoding the protein shown in SEQ ID No. 2) was constructed into the pGex-6p-1 vector plasmid, hlyA (as shown in SEQ ID No. 3, encoding the protein shown in SEQ ID No. 4) and flgJ (as shown in SEQ ID No. 5, encoding the protein shown in SEQ ID No. 6) were constructed into the pET-28a vector plasmid, and ndK (as shown in SEQ ID No. 7, encoding the protein shown in SEQ ID No. 8) was constructed into the pET-28a vector plasmid, as follows:

[0043] Based on the *L. intracellularis* genome (accession number: AM180252.1) from Genebank, amplification primers Omp2, hlyA, flgJ, and ndK were designed: Omp2 (SEQ ID No. 9, SEQ ID No. 10), hlyA (SEQ ID No. 11, SEQ ID No. 12), flgJ (SEQ ID No. 13, SEQ ID No. 14), and ndK (SEQ ID No. 15, SEQ ID No. 16). Using the nucleic acid of *L. intracellularis* isolate PHE-JS01 as a template, a 25 μL amplification system was established (12.5 μL 2×Primer STAR MaxDNA Polymerase, 9.5 μL dd...). PCR amplification was performed using H2O, 1 μL of upstream primer, 1 μL of downstream primer, and 1 μL of nucleic acid template (amplification program: 98℃ pre-denaturation for 5 min, 98℃ denaturation for 30 s, 55℃ annealing for 5 s, 72℃ extension for 20 s for a total of 30 cycles, and 72℃ final extension for 10 min).

[0044] Table 1 Primer sequences

[0045]

[0046]

[0047] After PCR amplification of the target gene, the fragments were purified by agarose gel electrophoresis using a gel recovery kit. The pGex-6p-1 plasmid and the Omp2 target fragment were double-digested with BamHI and SalI according to a double-enzyme digestion system (1 μL restriction enzyme 1, 1 μL restriction enzyme 2, 5 μL 10× Quick Cut Buffer, 1 μg DNA or plasmid, dd H2O to 50 μL). The pET-28a plasmid and the hlyA, flgJ, and ndK target fragments were double-digested with BamHI and XhoI at 37℃ for 2 h. The digestion products were recovered, and 4 μL of the recovered target fragment, 1 μL of the vector, and 5 μL of Ligation Mix were mixed and ligated overnight at 16℃.

[0048] The ligation products pGex-6p-1-Omp2, pET-28a-hlyA, pET-28a-flgJ, and pET-28a-ndK were transformed into competent DH5α cells using chemical transformation. Single colonies were selected and cultured in small quantities for identification. Subsequently, the constructed plasmids were transformed into competent BL21(DE3) cells. Results for positive strains are shown below. Figure 1 As shown, the sizes of pGex-6p-1-Omp2, pET-28a-hlyA, pET-28a-flgJ, and pET-28a-ndK are 891bp, 753bp, 681bp, and 417bp, respectively, which are in line with expectations. Sequencing of the positive strains and comparison of the sequencing results showed no premature termination or mutations in the protein sequences, indicating that the target fragment was successfully ligated into the expression vector and the expression strain was successfully constructed.

[0049] Example 2: Expression and purification of recombinant proteins

[0050] The expression cultures of pGex-6p-1-Omp2, pET-28a-hlyA, pET-28a-flgJ, and pET-28a-ndK were transferred at a ratio of 1:100 to 100 mg kanamycin in LB medium and cultured with shaking at 37°C until the bacterial culture showed OD values. 600nmWhen the pH value was 0.6–0.8, 100 μL of IPTG was added to a final concentration of 1 mM, and the cells were then transferred to a shaker at 16 °C for induction for 16 h. After induction, the bacteria were harvested, centrifuged at 12000 rpm for 10 min, washed with PBS, resuspended in 20 mL of PBS, and sonicated. The lysate was then centrifuged at 12000 rpm for 10 min, and the supernatant and precipitate were separated. The precipitate was resuspended in an equal volume of PBS. Samples of uninduced whole bacteria, induced whole bacteria, supernatant, and precipitate were collected and analyzed by SDS-PAGE to determine the expression of the target protein. Simultaneously, the inducer concentration and induction time were optimized to obtain the optimal protein expression conditions. The optimal induction conditions were 1 mM IPTG at 16 °C for 16 h.

[0051] The newly streaked recombinant strains were inoculated and transferred to 1 L LB liquid medium for expansion culture. Each recombinant strain was induced under its optimal induction conditions. After induction, the protein was expressed and purified in large quantities.

[0052] The supernatant collected after sonication was purified using conventional GST-tagged affinity chromatography and nickel-column affinity chromatography. The purified protein was then concentrated by ultrafiltration using a suitable ultrafiltration tube. The purified protein was filtered to 0.22 μm for sterilization. Finally, the purity of the purified recombinant protein was determined using a BCA kit and SDS-PAGE.

[0053] Omp2, hlyA, flgJ, and ndK proteins were all expressed in the supernatant, with expected sizes of 59 kDa, 28 kDa, 26 kDa, and 14 kDa, respectively. Protein purification results are shown below. Figure 2 As shown, the results were consistent with expectations, and the purified target proteins all had high purity, making them suitable for subsequent animal experiments.

[0054] Example 3: Preparation of porcine ileitis subunit vaccine

[0055] Purified Omp2, hlyA, flgJ, and ndK proteins were used as antigens to prepare a vaccine. The porcine ileitis subunit vaccine, after passing sterility testing, was mixed with ISA201VG adjuvant at a 1:1 (v / v) ratio to prepare the vaccine. Each dose of the vaccine contained 200 μg of each protein, and 0.01 M PBS buffer was used as a volume supplement solution for vaccine preparation.

[0056] Example 4: Evaluation of Immunoprotective Effects of Porcine Ileitis Subunit Vaccine

[0057] 1. Laboratory animals and grouping

[0058] Seventy-two 3-week-old piglets were randomly divided into 9 groups of 8 piglets each. Immunization was performed according to Table 2. The immunization route was intramuscular injection in the hind neck, with a dose of 2 mL per piglet. A booster immunization was administered on day 21 after the initial immunization, with the same dose as the initial immunization.

[0059] Table 2 Grouping of piglets for immunization and challenge

[0060]

[0061] 2. Preparation of virulent intracellular Lawsonia bacterial suspension

[0062] The virulent intracellular Lawsonia strain PHE-JS01, cryopreserved in DMSO, was rapidly thawed at 37°C and inoculated into a fresh McCoy cell monolayer. It was then cultured in a 37°C tri-gas culture system for 6 days, with the medium changed every 2 days. The harvested bacterial culture was stored at 4°C and the bacterial count was performed. A 5×10⁻⁶ DMEM solution was prepared. 7 A highly virulent intracellular Lawsonia bacterial solution containing copies / mL.

[0063] 3. Virus challenge experiment

[0064] 35 days after the initial vaccination, piglets were challenged with the virus via oral gavage according to Table 6, with a challenge dose of 1×10⁻⁶. 9 Copies / head, observed for 21 days. Blood was collected from the vena cava on days 0, 21, and 35 post-immunization, and again on day 21 post-challenge, and serum was separated and frozen for later use. Fecal samples were collected on days 0, 7, 14, and 21 post-challenge and nucleic acid was extracted for quantitative real-time PCR detection. On day 21 post-challenge, pigs were euthanized and dissected. The ileum was opened and gross lesion scoring was performed. Ileal tissue was collected, fixed, sectioned, stained with hematoxylin and eosin (HE), and subjected to intracellular Lawsonia solani-specific IHC detection and scoring.

[0065] 4. Determination of serum IgG antibody levels in piglets

[0066] A. Dilute intracellular Lawsonia subunit protein to 2.5 μg / mL with coating buffer, coat ELISA plates with 100 μL / well, and incubate overnight at 4°C.

[0067] B. Discard the coating solution, add 150 μL of PBST to each well, and wash three times, once every 3 minutes.

[0068] C. Add 200 μL of blocking solution containing 0.5% BSA to each well and incubate at 37°C for 2 hours.

[0069] D. Washing: Add 200 mL of PBST to each well and wash 3 times, 3-5 minutes each time.

[0070] E. Dilute the serum samples of each group of experimental piglets with blocking buffer to a dilution of 1:200, add them to the ELISA plate in the order of sample number, 100 μL / well, and incubate at 37°C in the dark for 1 hour.

[0071] F. Wash the plate by adding 200 μL of PBST to each well and washing three times for 3-5 minutes each time.

[0072] G. Dilute the enzyme-labeled secondary antibody, namely horseradish peroxidase (HRP)-labeled goat anti-pig IgG, 5000-fold with buffer, 100 μL / well, and incubate at 37°C for 1 hour in the dark.

[0073] H. Wash the plate by adding 200 mL of PBST to each well and washing three times for 3-5 minutes each time.

[0074] I. Color development: Use TMB substrate solution for color development, 100 μL per well, and develop for 10 minutes at room temperature in the dark.

[0075] J. Termination of reaction: Add 100 μL / well of stop solution (2 mol / L H2SO4 solution) and measure the OD value at 450 nm wavelength using an ELISA reader.

[0076] 5. Dynamic monitoring of fecal bacterial excretion

[0077] Feces were collected from piglets in each group at 0, 7, 14, and 21 days after challenge. Fecal DNA was extracted using a commercial DNA extraction kit, and the LI load in the feces was detected by real-time PCR.

[0078] 6. Gross lesion score

[0079] On day 21 after viral challenge, necropsy was performed on piglets in each group to observe ileal lesions and score them. The scoring criteria were as follows: no lesions 0 points, single thickened lesion 1 point, blocky thickened lesion 2 points, diffuse thickened lesion 3 points, and hemorrhagic thickened lesion 4 points.

[0080] 7. Microscopic lesions of the ileum

[0081] On day 21 post-infection, necropsy was performed. Ileal tissue was collected for fixation, sectioning, H&E staining, and immunohistochemical staining with intracellular Lawsonia solani-specific monoclonal antibodies, and then scored. The scoring criteria for microscopic lesions were: no lesion 0 points, single typical lesion 1 point, few (<30%) typical lesions 2 points, 30%-60% typical lesions 3 points, and many (60%) typical lesions 4 points. The scoring criteria for immunohistochemical staining were: no staining 0 points, single staining 1 point, few (<30%) staining 2 points, 30%-60% staining 3 points, and many (60%) staining 4 points.

[0082] 8. Experimental Results and Analysis

[0083] like Figure 3As shown, the antibody levels in piglet serum significantly increased after immunization, reaching their maximum 14 days after the second immunization. This indicates that the vaccine immunization group can induce humoral immunity in piglets. The vertical axis in the figure represents the sample value / negative value (P / N value, OD value of the immunized group / OD value of the blank control group), and the horizontal axis represents the detection time point.

[0084] like Figure 4 As shown in the figure, the viral shedding in piglets after vaccination was significantly lower than that in the positive control group, indicating that the vaccine-immunized group can reduce viral shedding in piglets. The vertical axis of the figure represents the logarithm of fecal bacterial copy number (LOG10 copies / g), and the horizontal axis represents the number of weeks after challenge.

[0085] As shown in Tables 3, 4, and 5, the gross lesions, microscopic lesions, and immunohistochemical scores of piglets were significantly reduced after immunization. Figure 5 As shown, the ileum tissue in the challenge control group was significantly thickened and generally accompanied by hemorrhage. H&E staining showed a significant increase in intestinal crypt epithelial cells and a large decrease in goblet cells. Immunohistochemistry showed a large number of bacteria within the intestinal crypt epithelial cells. The subunit vaccines in this experiment showed some resistance to Lawsonia intracellularis infection. As shown in Table 6, the Omp2+hlyA combination was the most effective, with a lesion reduction rate of 77.64%, followed by Omp2+ndK, with a lesion reduction rate of 74.12% (lesion reduction rate = (sum of gross lesions, microscopic lesions, and immunohistochemical scores in the control group - sum of gross lesions, microscopic lesions, and immunohistochemical scores in the immunized group) / sum of gross lesions, microscopic lesions, and immunohistochemical scores in the control group).

[0086] Table 3 Number of Gross Lesion Scores

[0087]

[0088] Table 4 Microscopic Lesion Scoring

[0089]

[0090]

[0091] Table 5 Immunohistochemical scores

[0092]

[0093] Table 6. Statistical Table of Lesion Reduction Rate Scores

[0094]

[0095]

[0096] This invention enables the soluble expression of Omp2, hlyA, flgJ, and ndK proteins.

[0097] This invention constructs a subunit vaccine using the water-in-oil-in-water adjuvant ISA201VG. To evaluate the vaccine's protective efficacy, an immune challenge protection experiment was conducted, including a protein immunization group, a challenge control group, and a blank control group. Results after challenge showed:

[0098] 1. Antibody level: The antibody level in the serum of piglets was significantly increased after immunization, reaching its highest level 14 days after the second immunization.

[0099] 2. Changes in viral shedding: After vaccination, the viral shedding of piglets was significantly reduced compared with the positive control group, with the most significant reductions observed in the Omp2 group, hlyA group, Omp2+hlyA group, and Omp2+ndK group.

[0100] 3. Reduction rate of lesions: The subunit vaccines in this experiment have a certain resistance to Lawsonia intracellularis infection. Among them, the Omp2+hlyA combination and the Omp2+ndK combination are the most effective, with lesion reduction rates of 77.64% and 74.12%, respectively.

[0101] The above results indicate that the porcine ileitis subunit vaccine, which combines prokaryotic Omp2 protein with hlyA, flgJ, and ndK proteins, can elicit a strong humoral immune response in piglets and has a certain effect on resisting Lawsonia intracellularis infection.

[0102] SEQ ID No. 1 (Omp2 nucleotide sequence)

[0103]

[0104] SEQ ID No.2 (Omp2 amino acid sequence)

[0105] MKIIHSAIFAVTLLTAWSTVCFAAEVTASCTKRVESYNYLVDYSGSMMMKHVAVREPKIELAKEAILKINAAMPKMSYQGGLYTFAPYSVIIPQGSWNSCVAECAVNTIKSDLEIFGRLTPMGDGIKMHETVINQMPPQAAVILLTDGHNNLGMNPVEEVKSIYQTNPNVCFHVVSFADDAEGKAIIDQIVALNSGSVLVDGLQLLQNPAVCQEFVNSVFCQEQILVTEEVVVLRGVNFAFDSFALDDTAKAILEETVRLIRANPDFNVRLLGWTDSTGPDAYNLRLSQERADAVKNFLVKMGIPSNRLFAKGMGKSYQYNNATKEGRYMNRRTELVFFD

[0106] SEQ ID No.3 (hlyA nucleotide sequence)

[0107] ATGGCCAAACATAAAGTACGTGCTGATGAACTTGTTTTTTTACAAGGGTTAGCAGAAAGTCGTGAACAAGCTAAACGACTTATTATGGCAGGTAAGGTTACATTAACTAATAATTCTACAACTATACCATTACGTTTGGAAAAACCAGGACATAAATATCCATTAGAAAGTATCTGCAGTTTAATAGGGGTAGAACGTTTTGTGAGTAGAGGAGCATATAAGCTATTAACTGCTCTAGATTTTTTTAAAATTGATGTAAAAAGTTGTATTTGTCTTGATGCAGGCGCATCTACTGGTGGGTTTACAGATTGTCTTTTACAACATGGAGCATCTAAAGTATATGCGATTGATGTAGGCAAAGGTCAATTACATGAGAAACTGTATACTAATGAACAAGTTATAAATATTGAGGGAGTGAATTTACGTACAGCATCTAAAGATCTTATTCCTGAAGAAGTAGATATTTTAACTATTGATGTTTCTTTTATATCGCTTACTTTGATTTTACCGTCATGTATACGTTGGCTAAAGGCTTCCGGAATTATTATTGCCTTAATAAAGCCTCAATTTGAATTATATCCAGATAAAATAAAAAAAGGTGTAGTAAAAGAAACTAGCTTGCAATATGAAGCAGTAGAAAAAATTATTCATTTTTGTCAATCAGAACTTGGACTTATATTTATTGGTGTTGTTCCGTCGGTAATAAAAGGTCCAAAAGGAAATCAAGAATATCTTATTTACTTGAAAAAACGT

[0108] SEQ ID No.4 (hlyA amino acid sequence)

[0109] MAKHKVRADELVFLQGLAESREQAKRLIMAGKVTLTNNSTTIPLRLEKPGHKYPLESICSLIGVERFVSRGAYKLLTALDFFKIDVKSCICLDAGASTGGFTDCLLQHGASKVYAIDVGKGQLHEKLYTNEQVINIEGVNLRTASKDLIPEEVDILTIDVSFISLTLILPSCIRWLKASGIIIALIKPQFELYPDKIKKGVVKETSLQYEAVEKIIHFCQSELGLIFIGVVPSVIKGPKGNQEYLIYLKKR

[0110] SEQ ID No.5 (flgJ nucleotide sequence)

[0111] ATGATTAATCCTATTGATCCTTCTTTAGCTCAAAATTCAGCTGAAGAACAAAACTTAAAAGAAATAAAACGTAAAGCAGATAAAGAAATACAAAATCTTGATCCAAATGCTGCTGCAGCTAAAGAAAAAAAACTTCGTGAAGCTGCAGAAGGATTTGAAGCTATCTTTATTCAACAAATGTGGCAAAGTATGCGTGCAAGCCTTCCTAAAGAAGGTATAATGCATAGTCGTGAAGAACAATTTTGGCAAGGGATGTACGATCAAGAGTTAGGCAAAAGTATGGCTTCAGCTGGAGGGATTGGACTTGCTGATATGATGATGGCTCAACTATCTAAAAAATTAGAAAATGCTAGTGAAGTTACAGCAGAAAGTATGCATCGAACACCACTAGATATAAAACCAGTTCCACTTATTGAAGAAAAAGCACCTCAAGAACTAAAACCTACTAATAAAAAACTAGAACATATCTATAATGGCGAATTAAGCCAACATAACTCTTCTATAAAAAATAATACAAAACCTTCTACAACTACTAACACACTTACACCTGTTGAAGAAGCTCTAAATGAATTTTCTAAGCAAATAAAAACACCTTCTTCAACACCTACAGAAAATATAACATCTTCAGACTCAAATACAAAACCTCCTATTATATCTAATACAGTTACTATCCCAAAAAAT <0000​​​​​

[0114] SEQ ID No. 7 (ndK nucleotide sequence)

[0115] ATGCAACACACATTTGCTCTAATTAAGCCTGACGCTGTTCAAAGAAATCTCATTGGTGCTATTATTAACATGATTGAAAAAAATGACTTTTATATTTCAGCTATGAAAATGTTACAAATGAATCGTCAACAAGCTGAAGGATTTTATAGTGTACACCGTGAACGCCCATTTTTTAATGAACTTGTTGATTACATGATCTCTGGCCCT ATTGTTAGTCTTATATTAACAGGAGAAAATGCTGTTACTCGCTATAGAGAACTAATGGGAGCAACAAATCCTCAAAATGCACAGGAAGGAACTATTCGAAAATCTTTTGCAATTAGTTTAATGGAAAATGCTGTCCATGGTTCTGATTCTGATGAAAATGCAATAATAGAGATTAATTATTTTTTTAATGCCTTTGAAAGGATACGG

[0116] SEQ ID No. 8 (ndK amino acid sequence)

[0117] MQHTFALIKPDAVQRNLIGAIINMIEKNDFYISAMKMLQMNRQQAEGFYSVHRERPFFNELVDYMISGPIVSLILTGENAVTRYRELMGATNPQNAQEGTIRKSFAISLMENAVHGSDSDENAIIEINYFFNAFERIR

[0118] The above embodiments are merely illustrative examples of specific implementations of the present invention and are not intended to limit the scope of the technical solutions of the present invention. Any reasonable modifications made by those skilled in the art to the technical solutions based on the technical concepts disclosed in the present invention, without departing from the core design principles of the present invention, through adjustments, optimizations, or equivalent substitutions, shall fall within the scope of protection claimed by the present invention.

Claims

1. Intracellular Lawsonia antigen protein, characterized in that, The antigen protein is selected from hlyA protein, flgJ protein, and ndK protein; the amino acid sequence of hlyA protein is shown in SEQ ID No. 4; the amino acid sequence of flgJ protein is shown in SEQ ID No. 6; and the amino acid sequence of ndK protein is shown in SEQ ID No.

8.

2. The gene encoding the intracellular Lawsonia antigen protein of claim 1, characterized in that, The nucleotide sequence of the gene encoding the hlyA protein is shown in SEQ ID No. 3, the nucleotide sequence of the gene encoding the flgJ protein is shown in SEQ ID No. 5, and the nucleotide sequence of the gene encoding the ndK protein is shown in SEQ ID No.

7.

3. The method for preparing the intracellular Lawsonia antigen protein according to claim 1, characterized in that, hlyA, flgJ, and ndK proteins were all obtained through prokaryotic expression.

4. The preparation method according to claim 3 is characterized in that, Includes the following steps: (1) Construction of recombinant plasmids: The hlyA, flgJ and ndK genes were inserted between the BamHI and XhoI restriction sites of the prokaryotic expression vector pET-28a, respectively, and transformed into DH5α competent cells. Positive clones were screened, plasmids of positive clones were extracted and sequenced, and plasmids with the correct target fragments were selected and transformed into BL21(DE3) competent cells. (2) Recombinant protein expression and purification: positive clones are cultured, protein expression is induced, and the protein is purified by affinity chromatography.

5. A porcine ileitis subunit vaccine, characterized in that, The porcine ileitis subunit vaccine uses one or more of the hlyA protein, flgJ protein, and ndK protein as antigens as described in claim 1, or uses a combination of one or more of the hlyA protein, flgJ protein, and ndK protein with the Omp2 protein as antigens.

6. The porcine ileitis subunit vaccine according to claim 5, characterized in that, The amino acid sequence of the Omp2 protein is shown in SEQ ID No. 2, and the nucleotide sequence of the gene encoding the Omp2 protein is shown in SEQ ID No.

1.

7. The porcine ileitis subunit vaccine according to claim 5, characterized in that, The porcine ileitis subunit vaccine also includes an adjuvant, wherein the adjuvant is a water-in-oil-in-water adjuvant, preferably ISA201VG.

8. The porcine ileitis subunit vaccine according to claim 7, characterized in that, The volume ratio of antigen to adjuvant is 1:

1.

9. The use of the intracellular Lawsonia antigen protein of claim 1 in the preparation of a subunit vaccine for the prevention of porcine ileitis.

10. The use of the gene encoding intracellular Lawsonia antigen protein as described in claim 2 in the preparation of a subunit vaccine for the prevention of porcine ileitis.

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