Three candidate antigens of Actinobacillus pleuropneumoniae and their applications

Abstract

This invention discloses three candidate antigens of Actinobacillus pleuropneumoniae (APP) and their applications in the preparation of APP detection kits and subunit vaccines. The applicant screened APP antigen proteins using a combination of bacterial surface proteomics, reverse vaccinology, phage display libraries, immunoprecipitation, and next-generation high-throughput sequencing technologies, obtaining three candidate antigens (PotA, SRP, and FtsN). These three antigens exhibit high reactivity and immunogenicity, and can be used for the clinical diagnosis and treatment of APP. The research results of this invention are of great significance for the detection and control of Actinobacillus pleuropneumoniae.

Description

[0001] This application is a divisional application of the following patent application:

[0002] The original application was filed on April 21, 2023.

[0003] Application Number: 202310436919.4

[0004] Invention Title: Three Candidate Antigens of Actinobacillus pleuropneumoniae and Their Applications Technical Field

[0005] This invention belongs to the field of animal infectious disease prevention and control and biotechnology, specifically involving three candidate antigens of Actinobacillus pleuropneumoniae and their applications in clinical diagnosis and vaccine development. Background Technology

[0006] Porcine contagious pleuropneumonia is a highly contagious, contact-transmitted respiratory disease in pigs caused by Actinobacillus pleuropneumoniae (APP). Its main clinical features are acute hemorrhagic, fibrinous pneumonia and chronic fibrinous, necrotic pleuropneumonia. Due to its high morbidity and mortality rates, reduced average daily weight gain and feed conversion ratio, slaughter losses, and intervention costs, this disease has a significant impact on animal welfare and economic production.

[0007] Accurate diagnosis of pleuropneumonia is crucial for the prevention and control of this disease. Although some commercial kits based on the toxin ApxIV are available for detecting APP, their high cost and limited sample capacity significantly restrict their application. Identifying new candidate antigens for detecting clinical APP infection would allow for large-scale testing of samples after a single purification process, overcoming limitations imposed by kit cost and sample size, which is vital for accurate diagnosis of the disease.

[0008] Vaccination is a crucial means of preventing pleuropneumonia. However, with 19 serotypes of APP, developing an effective APP vaccine remains a significant challenge. Trivalent inactivated vaccines based on serotypes 1, 2, and 7 provide effective protection against homologous strains but are less effective against heterologous strains. Some attenuated mutant live vaccines lacking the APP toxin offer cross-protection against both homologous and heterologous strains, but their safety and efficacy still need improvement. Overcoming these limitations involves developing multivalent subunit vaccines using antigens that are highly conserved across different serotypes to provide protection against multiple serotypes and even multiple pathogens. Researchers have already developed commercially available APP subunit vaccines, PleuroStar APP and [other vaccines], based on toxin proteins and outer membrane proteins. APP provides good protection against certain serotypes of infection. However, these commercially available subunit vaccines are costly, necessitating research into new processes and methods, continuous search for new vaccine targets, cost reduction and efficiency improvement, and enhanced product quality.

[0009] Therefore, identifying new candidate antigens for Actinobacillus pleuropneumoniae will be of great significance for clinical diagnosis and vaccine development. Summary of the Invention

[0010] The purpose of this invention is to provide new APP candidate antigens to provide targets for the development of novel vaccines and diagnostic agents.

[0011] The technical solution of the present invention is as follows:

[0012] The applicant first identified several surface and secretory proteins from *Acinetobacter aubergine* (APP) using bacterial surface proteomics and reverse vaccinology. These proteins were then truncated along with other known proteins. Primers were designed for these truncated fragments, and PCR amplification was performed using APP genomic DNA as a template. The amplified products were double-digested with enzymes, ligated to phage vector arms, and packaged in vitro to construct a bacterial antigen phage display library. Next, the applicant used immunoprecipitation and next-generation high-throughput sequencing to screen for high-throughput immunogenic antigens at the whole-genome level of APP bacteria, identifying 16 APP antigens that could bind to serum. Finally, through serological reaction and immunogenicity verification, three of these antigens were further screened and found to possess both serological reactivity and immunoprotective efficacy.

[0013] These three antigens are PotA, SRP, and FtsN proteins, whose functions have been annotated. They are spermine / putrescine ABC transporter ATP-binding proteins, signal recognition particles, essential cell division proteins for activating spacer peptidoglycan synthesis and cell contraction, respectively. Their amino acid sequences are shown in SEQ ID NO:1-3.

[0014] The applicant named the genes encoding the above three proteins HBS1_10, HBS1_15 and HBS1_16 respectively, and their nucleotide sequences are shown in SEQ ID NO:4-6 respectively.

[0015] Then, the proteins were expressed and purified using an E. coli prokaryotic expression system, and three proteins were successfully obtained.

[0016] Three proteins were obtained and used as coating antigens. Enzyme-linked immunosorbent assay (ELISA) showed that they could distinguish between APP-positive and APP-negative sera, consistent with the results of commercially available diagnostic kits. Homology analysis with proteins from different bacterial species revealed low homology between the FtsN protein and other bacteria. Furthermore, ELISA showed low cross-reactivity of the three proteins with positive sera from other pathogens, indicating that they can serve as diagnostic targets for specific detection of APP.

[0017] Homology analysis of the three proteins with proteins from different serotypes of APP revealed that they were highly conserved across 12 different APP serotypes. Immunization of animals with the three proteins mixed with adjuvants, using ELISA, showed that all three proteins induced high levels of humoral immune responses, with a preference for inducing Th2-type immune responses. The three selected proteins, PotA, SRP, and FtsN, provided immune protection in mice, thus offering novel candidate antigens for the development of APP subunit vaccines.

[0018] In summary, the three proteins obtained by this invention, or the gene encoding any one of the proteins, or the expression vector containing any one of the genes, or the host bacteria containing any one of the expression vectors, can all be used for the detection and immunotherapy of APP.

[0019] The present invention also provides a specific embodiment of indirect ELISA detection of APP antibody using the protein described herein. Of course, the embodiments of the present invention are not limited thereto. For example, antibodies can also be prepared using the protein or gene provided by the present invention, and the antibodies can be used to perform direct ELISA detection of APP antigen.

[0020] The present invention also provides a specific embodiment of preparing a subunit vaccine using the protein and providing immune protection to animals.

[0021] The present invention has the following advantages:

[0022] 1. This invention is the first to use surface proteomics and reverse vaccinology, combined with phage display libraries, immunoprecipitation, and next-generation sequencing technologies to screen APP antigen proteins. This screening is a high-throughput, unbiased, and low-cost screening performed at the bacterial whole genome level, and the results have high accuracy, setting an example for the screening of other bacterial antigen proteins.

[0023] 2. This invention screens out three APP antigen proteins in one step. After verification, these three proteins have high reactivity and immunogenicity and can be used for the clinical diagnosis and treatment of APP. This invention provides three new antigen targets for this purpose. Attached Figure Description

[0024] Figure 1Agarose gel electrophoresis results of PCR products of the three target genes. M is a 2kb DNA marker; 10, 15 and 16 represent the PCR-amplified fragments HBS1_10, HBS1_15 and HBS1_16, respectively.

[0025] Figure 2 PCR identification results of three target gene expression plasmids. Two DNA markers of 2kb and 5kb were used; 10-1 / -2, 15-1 / -2 and 16-1 / -2 represent the three plasmids identified by colony PCR.

[0026] Figure 3 SDS-PAGE electrophoresis bands of three purified proteins. MW represents the high molecular weight protein marker.

[0027] Figure 4 ELISA results of three recombinant proteins in APP-infected serum. AC represents the detection of APP infection in porcine serum coated with PotA, SRP, and FtsN proteins, respectively; D represents the detection results of a commercially available kit.

[0028] Figure 5 Homology analysis of the three proteins with proteins from other bacterial species.

[0029] Figure 6 The reactivity of three proteins with porcine sera containing Streptococcus suis (A) and Haemophilus parasuis (B).

[0030] Figure 7 Homology analysis of three proteins with proteins derived from 12 other different serotypes of Actinobacillus pleuropneumoniae.

[0031] Figure 8 Animal immunization flowchart.

[0032] Figure 9 Detection of IgG antibody levels after immunization with three proteins.

[0033] Figure 10 Indirect ELISA was used to detect antibody titers of IgG1 and IgG2a and to analyze IgG subtypes.

[0034] Figure 11 Survival rate of mice immunized with the three proteins after challenge. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0036] The applicant previously used a variety of technologies, including phage display libraries, immunoprecipitation, and next-generation high-throughput sequencing, to establish a method for unbiased, high-throughput screening of immunogenic antigen proteins at the bacterial whole-genome level. This method has been filed for another Chinese invention patent prior to this patent application, with patent application number 202310239675.0 and titled "A method for high-throughput screening of immunogenic antigen proteins at the bacterial whole-genome level".

[0037] Using the methods described above, the applicant screened multiple immunogenic antigen proteins from the genome of Streptococcus suis. However, for clinically isolated bacteria or those with limited known genomic sequences, such as APP, it is difficult to screen for antigens by identifying core genes. Therefore, the applicant first identified 333 surface proteins of APP using a combination of biotinylate labeling, trypsin digestion, streptavidin enrichment, and mass spectrometry (i.e., surface proteomics). Then, 129 secretory proteins of APP were identified using reverse vaccinology. These surface proteins, secretory proteins, and seven proteins reported in the literature were then fragmented into 200-amino acid fragments, with each fragment overlapping by 100 amino acids, resulting in a total of 1359 fragments. These fragments were then amplified by PCR, and the amplification products were mixed in equal volumes and double-digested with enzymes. Subsequently, they were ligated to the T7 10-3b arm of a digested phage vector, and the ligation product was packaged in vitro to form an infectious phage library. Finally, high-throughput sequencing analysis of the proliferated phage library revealed that more than 80% of the fragments were present in the library, indicating that the library was successfully constructed.

[0038] To facilitate subsequent phage immunoprecipitation sequencing analysis, the applicant prepared convalescent serum from naturally infected mice. The serum was co-incubated with a phage library. After incubation, protein A / G magnetic beads were added for further incubation. Unbound phages were washed away, and the mixture was boiled with ddH2O to release the phages bound to the serum. Finally, two rounds of PCR amplification were performed. The products from the second round of PCR amplification were recovered and analyzed using high-throughput sequencing to screen for significantly enriched fragments.

[0039] The applicant screened 16 serum-binding APP antigens using bacterial surface proteomics, reverse vaccinology, and BacScan technology, naming them HBS1_01, HBS1_02, ..., and HBS1_16. Among them, three antigens, PotA (HBS1_10), SRP (HBS1_15), and FtsN (HBS1_16), were confirmed to have the ability to react with serum and provide immunoprotective effects.

[0040] PotA, the spermidine / putrescine ABC transporter ATP-binding protein, is part of the PotABCD ABC transporter complex and participates in the introduction of spermidine / putrescine, responsible for the energy coupling of the transport system. Signal recognition particles (SRPs) are ribonucleoprotein particles used to target proteins containing signal peptides to the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. FtsN is an essential cell division protein that activates spacer peptidoglycan synthesis and cell contraction, acting on both sides of the membrane through interactions with FtsA in the cytoplasm and with the FtsQBL complex in the periplasm. These three proteins, or their homologous family proteins, have not been reported to have immunoprotective effects or potential as diagnostic targets. Therefore, using the *Actinobacillus pleuropneumoniae* strain APP HBS1 isolated in our laboratory as a template, the applicant designed primers, cloned the three genes, and expressed the three proteins they encode. Serological testing revealed that all three proteins have the potential to detect *Actinobacillus pleuropneumoniae*. Animal experiments revealed that all three proteins could induce high levels of humoral immune responses and provide partial immune protection in mice, thus providing new candidate targets for the development of Actinobacillus pleuropneumoniae subunit vaccines.

[0041] Key material sources and explanations:

[0042] Actinobacillus pleuropneumoniae APP HBS1 strain: Actinobacillus pleuropneumoniae serotype 1 HBS1 strain was isolated and preserved in our laboratory. The whole genome sequence has been uploaded to the NCBI database, GenBank Accession number: CP115971.

[0043] pET32a vector: modified and preserved by our laboratory.

[0044] Escherichia coli BL21(DE3)RIPL competent cells: purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0045] Balb / c mice: purchased from the Experimental Animal Center of Huazhong Agricultural University.

[0046] Sequence list description:

[0047] SEQ ID NO: 1: Amino acid sequence of PotA protein from Actinobacillus pleuropneumoniae;

[0048] SEQ ID NO: 2: Amino acid sequence of SRP protein from Actinobacillus pleuropneumoniae;

[0049] SEQ ID NO: 3: Amino acid sequence of Actinobacillus pleuropneumoniae FtsN protein;

[0050] SEQ ID NO: 4: Nucleotide sequence of the PotA protein encoding gene (HBS1_10) of Actinobacillus pleuropneumoniae;

[0051] SEQ ID NO: 5: Nucleotide sequence of the gene encoding the SRP protein of Actinobacillus pleuropneumoniae (HBS1_15);

[0052] SEQ ID NO: 6: Nucleotide sequence of the gene encoding the FtsN protein of Actinobacillus pleuropneumoniae (HBS1_16);

[0053] SEQ ID NO: 7-8: Upstream and downstream primer sequences for amplifying the HBS1_10 gene;

[0054] SEQ ID NO: 9-10: These are the upstream and downstream primer sequences for amplifying the HBS1_15 gene;

[0055] SEQ ID NO: 11-12: These are the upstream and downstream primer sequences for amplifying the HBS1_16 gene.

[0056] Example 1: Preparation of recombinant protein

[0057] 1.1 PCR amplification of the target fragment

[0058] First, the transmembrane region and signal peptide of the amino acid sequences of the three proteins were predicted. Then, the nucleotide sequences corresponding to the amino acids of the three proteins were found, the signal peptide sequences were removed, and the regions located outside the cell membrane were selected. Three pairs of primers were designed for each protein. The primers included protective bases, restriction enzyme sites, and nucleotide sequences that bind to the template. The designed primers were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis.

[0059] Amplification of the APP HBS1_10 gene fragment: upstream primer 10F: 5'-CGC GGATCC ATGGAACAGTTAGAACA-3' (SEQ ID NO: 7; underlined restriction enzyme site BamHI); downstream primer 10R: 5'-CCG CTCGAG GCAATCTTCATCGTTGAGTA-3' (SEQ ID NO: 8; underlined is the restriction enzyme site XhoI).

[0060] Amplification of the APP HBS1_15 gene fragment: upstream primer 15F: 5'-CGC GGATCC ATGTTTGAAAACTTATCCG-3' (SEQ ID NO: 9; underlined restriction enzyme site BamHI); downstream primer 15R: 5'-CCG CTCGAG GCGTTTACCGAACATATTG-3' (SEQ ID NO: 10; underlined is the restriction enzyme site XhoI).

[0061] Amplification of the APP HBS1_16 gene fragment: upstream primer 16F: 5'-CGC GGATCC GAAAATGCGCCCGCACCG-3' (SEQ ID NO: 11; underlined restriction enzyme site BamHI); downstream primer 16R: 5'-CCG CTCGAG CATCGAAATAATTACACA-3' (SEQ ID NO: 12; underlined is the restriction enzyme site XhoI).

[0062] Single colonies of *Actinomyces pleuropneumoniae* were picked and incubated overnight at 37°C in TSB liquid medium containing 5% FBS and 0.1‰ NAD. The genome of the APP was extracted according to the manufacturer's instructions. Using the genomic DNA as a template, fragments 10, 15, and 16 of *Actinomyces pleuropneumoniae* were amplified by PCR, with amplification product lengths of 1131 bp, 1408 bp, and 820 bp, respectively.

[0063] The PCR amplification system is as follows: 1 μL template DNA, 10 μL high-fidelity enzyme, 1 μL each of forward and reverse primers, and 8 μL ultrapure water. After mixing thoroughly, set the PCR amplification program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 50-60℃ annealing for 15 s, 72℃ extension for 15-30 s, and 72℃ final extension for 5 min. Repeat the denaturation to extension process 30 times. Store at 16℃ for later use.

[0064] 1.2 Construction of expression plasmids

[0065] The PCR products were analyzed by agarose gel electrophoresis. Figure 1 The target fragment was cut at the appropriate location, and the amplified products of the three genes were recovered using a kit. These products were then double-digested with BamHI and XhoI, respectively. Simultaneously, the empty pET32a vector was double-digested with the same enzymes. The digestion reaction system was as follows: 5 μL of 10x rapid digestion enzyme buffer, 2.5 μL of BamHI, 2.5 μL of XhoI, 2 μg of the target fragment or vector, and ddH2O to a final volume of 50 μL. Digestion was performed at 37℃ for 30-60 min. The digestion products were analyzed by agarose gel electrophoresis, and the DNA of the cut target fragment was recovered and its concentration determined.

[0066] The digested products were ligated to the pET32a vector using T4 ligase. The ligation reaction mixture was as follows: 1 μL of 10x buffer, 0.5 μL of T4 ligase, and the required volume was calculated based on a 3:1 molar ratio of digested fragment to digested vector. ddH2O was added to bring the volume to 10 μL. The control group received no digested fragment, but was treated the same as the experimental group. Ligation was performed overnight at 16°C. The ligation products were then transformed into E. coli DH5α competent cells, following the steps below:

[0067] 1) Take 4 DH5α competent cells from the -80℃ freezer and thaw them on ice for 3-5 minutes. Add 3 5μL ligation products to 3 50μL competent cells respectively. Add the product without enzyme digestion fragment to the last competent cell and incubate on ice for 30 minutes.

[0068] 2) Turn on the water bath in advance and set it to 42°C. After the ice bath, place the competent state with the ligation product in the 42°C water bath for 90 seconds of heat shock, and immediately put it on ice for 2 minutes of ice bath.

[0069] 3) Add 500 μL of sterile LB liquid culture medium to each tube and incubate on a shaker at 37°C for 45-60 min;

[0070] 4) After incubation, centrifuge at 5,000 rpm / min for 3 min at room temperature, discard 450 μL of supernatant, resuspend the bacterial cells in the remaining liquid, add the bacterial solution to a plate containing ampicillin resistance, spread evenly with a spreader, and incubate upside down at 37°C overnight.

[0071] Comparing the colonies on the four plates, it was found that the control plate had only a few single colonies, indicating that the vector was completely digested with the double enzymes and no self-ligation or recombination occurred. In contrast, the three experimental groups all produced hundreds of single colonies. Two clones were randomly selected from each plate for PCR identification, and the results showed that the size of the colonies identified by PCR was the same as expected. Figure 2 Six plasmids were extracted using the mini-plasmid extraction kit and sent to the company for sequencing. The sequencing results and plasmid maps were analyzed by BLAST, and all six plasmids were found to be correctly sequenced. One plasmid from each plasmid was taken and named as pET32a-HBS1_10, pET32a-HBS1_15, and pET32a-HBS1_16, respectively.

[0072] 1.3 Protein Expression and Purification

[0073] The three recombinant plasmids were transformed into E. coli BL21(DE3)RIPL competent cells, respectively. The transformation steps were the same as in step 1.2, except that the heat shock time was changed to 45 s. Finally, the plasmids were plated on double antibiotic plates containing ampicillin and chloramphenicol and incubated overnight at 37°C with the plates inverted.

[0074] Positive clones were picked and cultured overnight at 37°C in LB liquid medium containing ampicillin and chloramphenicol, respectively. The next day, they were transferred to fresh LB medium at a ratio of 1:100 and cultured at 37°C for 2.5-3 hours until the OD value reached approximately 0.6. The shaker temperature was lowered to 30°C beforehand, and IPTG was added to a final concentration of 1 mM. The mixture was induced at 30°C for 4 hours. The cells were collected by centrifugation at 7,000 rpm / min for 10 minutes and washed three times with PBS. The cells were then resuspended in binding buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, adjusted to pH 8.0 with concentrated hydrochloric acid). The bacterial suspension was then subjected to low-temperature high-pressure disruption, centrifuged at 34,000 x g for 30 minutes, and the supernatant was collected. After filtration through a 0.22 μm pore size filter, the supernatant was bound to Ni-NTA His resin by peristalsis using a sample pump and then mixed with washing buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, adjusted to pH 8.0 with concentrated hydrochloric acid). Wash for about 20 minutes with NaCl, 20mM imidazole, and concentrated hydrochloric acid to adjust the pH to 8.0 to remove unbound proteins. Finally, elute the proteins bound to the His column with Elution buffer (50mM Tris, 300mM NaCl, 400mM imidazole, and concentrated hydrochloric acid to adjust the pH to 8.0).

[0075] Concentrate approximately 5 mL of the eluent through an ultrafiltration tube to replace the high concentrations of salt and imidazole. When selecting an ultrafiltration tube to concentrate the protein, generally choose one about one-third the size of the protein. For example, if the protein is 30–40 kDa, a 10 kDa ultrafiltration tube can be selected. Before use, wash the ultrafiltration tube twice with ultrapure centrifugation. Add the protein to the ultrafiltration tube and centrifuge at 4°C and 4,000 rpm. The centrifugation time depends on the specific protein. After each centrifugation, add a certain volume of GE buffer (20 mM Tris, 100 mM NaCl, adjusted to pH 8.0 with concentrated hydrochloric acid) to replace the high concentrations of salt and imidazole in the protein. After approximately 4–5 centrifugation replacements, the volume after centrifugation will be about one-tenth of the original volume, approximately 500 μL. Aspirate the protein from the ultrafiltration tube and transfer it to 1.5 mL centrifuge tubes, aliquoting 50 μL per tube. Store the aliquots at -80°C. 10 μL of purified protein was added to 2.5 μL of 5x SDS sample buffer and boiled for 5 min. SDS-PAGE electrophoresis was then performed at 80 V for 30 min and then at 120 V for 90–120 min. After electrophoresis, Coomassie Brilliant Blue staining was performed for 1 h, followed by destaining. Results showed that all three full-length proteins exhibited the target bands at the predicted locations, and the protein bands were clear with no obvious degradation. Figure 3 This indicates that three proteins have been successfully purified.

[0076] The three purified proteins were quantified using BSA. 5 mg of BSA was weighed and added to 5 mL of PBS. After mixing, 40 μL of 1 μg / μL BSA was added to 10 μL of protein loading buffer. 10 μL of each of the three purified proteins was then added to 2.5 μL of protein loading buffer, and the mixture was boiled for 5 min. The BSA loading volumes were 1.25 μL, 2.5 μL, 3.75 μL, 5 μL, and 6.25 μL, corresponding to 1–5 μg of BSA protein, respectively. SDS-PAGE electrophoresis was performed on the three proteins at loading volumes of 1.25 μL, 2.5 μL, and 3.75 μL. After electrophoresis, Coomassie Brilliant Blue staining and destaining were performed until the background was clear. Images were acquired using a gel imaging system, and IPWIN analysis was performed to determine the IOD values ​​of BSA and the target protein. A standard curve was plotted between the IOD value of BSA and the protein amount, and the concentrations of PotA, SRP, and FtsN were calculated to be 2.3 μg / μL, 4.2 μg / μL, and 3 μg / μL, respectively.

[0077] Example 2: Identification of Clinical Diagnostic Targets

[0078] 1.1 ELISA detection of Actinobacillus pleuropneumoniae infection in porcine serum

[0079] Three proteins used for serum screening were quantified and diluted to 2 μg / mL with coating buffer. 200 ng of protein was added to each well of the ELISA plate, and the plate was incubated overnight at 4°C. After blocking with 3% BSA at 37°C for 1 h, the blocking buffer was discarded, and the plate was washed 5 times. Porcine serum diluted 1:400 was added to each well, and the plate was blocked at 37°C for 1 h. Then, HRP-labeled goat anti-porcine IgG (1:6,000 dilution) was added, and the plate was blocked at 37°C for 1 h. The secondary antibody was discarded, and 100 μL of TMB chromogenic buffer was added. The plate was incubated at room temperature in the dark for 10 min. Finally, 100 μL of stop solution was added to terminate the reaction. The OD of each well was measured using an ELISA reader. 450 The absorbance value was measured. The procedure for detecting swine serum using the commercial Actinobacillus pleuropneumoniae toxin IV detection kit was followed according to the instructions. Results showed that out of 26 swine serum samples, 16 were positive for Actinobacillus pleuropneumoniae, and 10 were negative. These results were consistent with those of the commercial kit. Figure 4 This indicates that all 16 swine serum samples were infected with Actinobacillus pleuropneumoniae, and also shows that the three selected proteins specifically bound to the 16 positive swine serum samples.

[0080] 1.2 Homology analysis of candidate antigens

[0081] To further analyze whether all three proteins could serve as specific targets for detecting *Actinobacillus pleuropneumoniae*, homology analysis was performed on the three proteins with proteins from other bacterial species. Taking one protein as an example, the amino acid sequence of the protein was first pasted into the "Enter accession number(s), gi(s), or FASTA sequence(s)" box in NCBI protein blast. The "Database" was set to "Non-redundant protein sequences(nr)". *Actinobacillus* (taxid: 713) was removed from the "Organism Optional" section, and *Escherichia coli* (taxid: 562), *Glaesserella parasuis* (taxid: 738), *Streptococcus suis* (taxid: 1307), and *Pasteurella multocida* (taxid: 747) were selected for BLAST analysis. Only one representative bacterium from each species was selected for homology analysis. The homology results were displayed as a heatmap, with the vertical axis representing the four different bacteria and the horizontal axis representing the three candidate proteins from *Actinobacillus pleuropneumoniae*. The results showed that PotA and SRP had high homology with proteins derived from Haemophilus parasuis, Pasteurella multocida, and Escherichia coli, but low homology with proteins derived from Streptococcus suis. FtsN protein showed no detectable homology with proteins derived from Streptococcus suis, and low homology with proteins from the other three different bacteria. Figure 5 This suggests that the FtsN protein may serve as a target for the specific detection of APP.

[0082] 1.3 Detection of cross-reactivity with positive porcine sera from other pathogens

[0083] Evidence for determining specific targets for Actinobacillus pleuropneumoniae solely based on homology comparison results is insufficient. To preliminarily apply three proteins as targets for specific detection of Actinobacillus pleuropneumoniae, these three proteins were used as coating antigens, and their reactivity with positive sera from pigs containing Streptococcus suis and Haemophilus parasuis was detected by indirect ELISA. The results showed that PotA, SRP, and FtsN proteins did not cross-react with positive sera from pigs containing Streptococcus suis and Haemophilus parasuis. Figure 6 This indicates that all three proteins can serve as candidate targets for APP detection, but based on the analysis of homology results with other bacteria, FtsN is more suitable as a target for specific detection of APP.

[0084] Example 3: Evaluation of the immunoprotective effect of candidate antigens

[0085] 1.1 Homology analysis of three candidate antigens

[0086] To develop a cross-protective *Actinomyces pleuropneumoniae* vaccine, homology analysis was performed on three proteins with proteins derived from 12 other serotypes of *Actinomyces pleuropneumoniae*. First, the complete genome sequences of 12 pre-sequentially sequenced and assembled *Actinomyces pleuropneumoniae* were downloaded from the NCBI database. Homologous proteins of the three proteins were identified from each genome sequence. On the NCBI BLAST page, the "Align two or more sequences" option was selected. The amino acid sequence of one HBS1 protein was pasted into the upper box, and the homologous proteins from the 12 different serotypes were pasted into the lower box. BLAST analysis was then performed. Comparative analysis revealed that the homology of the three proteins across various serotypes was between 99% and 100%, indicating high conservation. Figure 7 ).

[0087] 1.2 ELISA detection of humoral immune responses to antigens

[0088] Twenty mice were randomly divided into four groups: three experimental groups and one control group, with five mice in each group. Female Balb / c mice were immunized with three proteins, 50 μg per mouse. The volume of each protein was calculated based on the immunization dose and protein concentration, and then emulsified completely with an equal volume of mineral oil adjuvant ISA 201 and injected intramuscularly. Control mice were immunized with an equal volume of PBS mixed with the adjuvant. Immunization was repeated every two weeks for a total of two immunizations. Figure 8 ).

[0089] Two weeks after the second immunization, blood was collected from the tail vein of mice and serum was separated. Total IgG antibody levels were detected using an indirect ELISA method. After blocking, the serum was serially diluted 1:400 to 18 wells and incubated at 37°C for 1 hour. HRP-labeled goat anti-mouse IgG (1:8,000 dilution) was added as the secondary antibody. The remaining steps were the same as the ELISA steps in 1.1 of Example 2. The results showed that, compared with the control group, FtsN protein induced the highest IgG antibody level, followed by SRP. Although PotA induced a lower IgG antibody level than the first two proteins, the endpoint titer for each protein was as high as 10. 6 ( Figure 9 This indicates that all three proteins induced a high level of humoral immune response.

[0090] To analyze IgG subtypes, antibody titers of IgG1 and IgG2a were detected by indirect ELISA using three proteins as coating antigens. The secondary antibody was replaced with HRP-labeled goat anti-mouse IgG1 and IgG2a (1:3,000 dilution), with the remaining steps the same. Results showed that, compared to the control group, the mixture of the three proteins with adjuvant induced higher levels of IgG1 antibody than IgG2 antibody. Figure 10A, B). Using 1 as the standard, the levels of IgG1 and IgG2a induced by the control group were comparable, while the IgG2a / IgG1 ratio of the three proteins was less than 1, indicating that the mixture of these three proteins with the adjuvant induced high levels of IgG1 antibody (A, B). Figure 10 C). IgG1 antibodies are associated with Th2-type responses, meaning that when these three proteins are mixed with adjuvants, they induce a Th2-type immune response.

[0091] 1.3 Challenge test to evaluate the immunoprotective effect of the three proteins on mice

[0092] Two weeks after the second immunization, all mice were challenged intraperitoneally with serotype 1 HBS1 at a dose of 2.5 x 10⁻⁶. 7 CFU. Mice were observed for 7 consecutive days after challenge. Results showed that 6 hours post-challenge, mice in all groups exhibited ruffled fur, huddling together, and decreased appetite. 12 hours post-challenge, all mice in the control group died, while mice in the PotA, SRP, and FtsN groups, although showing poorer mental state, all survived. Figure 11 24 hours after challenge, two mice in the SRP group survived, while one mouse each survived in the PotA and FtsN groups. The challenge results indicate that the neoantigens PotA, SRP, and FtsN proteins can all provide partial immune protection in mice. Subsequently, these antigens can be delivered to the body's immune system via specific antigen delivery systems to induce a highly efficient immune response, thereby enhancing the immunoprotective effect of individual proteins, or they can be used as components of subunit vaccines. In conclusion, these three antigens show promise as important candidate targets for the development of APP vaccines.

Claims

1. The use of the protein shown in SEQ ID NO:3 or its encoding gene, or an expression vector or host bacterium containing the encoding gene, in the preparation of a detection kit for Actinobacillus pleuropneumoniae (APP).

2. The application as described in claim 1, characterized in that: The kit is an indirect ELISA kit for detecting antibodies against Actinobacillus pleuropneumoniae, and the kit contains the protein described in claim 1.

3. The use of the protein shown in SEQ ID NO:3 or its encoding gene, or an expression vector or host bacterium containing the encoding gene, in the preparation of Actinobacillus pleuropneumoniae subunit vaccines.

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