Multi-epitope fusion protein targeting multiple serotypes of Gramseria parasuis antigens and its application

Abstract

The present invention belongs to the field of animal vaccines, and specifically relates to a multi-epitope fusion protein of Gramseria parasuis antigens targeting multiple serotypes and its application. The applicant conducted proteomic analysis on four clinical isolates of Gramseria parasuis serotypes 4, 5, and 13 with high virulence and good immunogenicity, and screened out 8 antigenic proteins. The 8 screened antigenic proteins were further analyzed, and finally an immunogenic multi-epitope fusion protein of antigens that can recognize the three serotypes was obtained. As a vaccine, the fusion protein achieved a 100% protection rate against piglets infected with the clinically isolated serotype 4 strain HB04, the serotype 5 strain SJZ05, and the serotype 13 strain GD20.

Description

Technical Field

[0001] The present invention belongs to the field of animal vaccines, and in particular relates to a multi-epitope fusion protein targeting Gramseria parasuis antigens of multiple serotypes and its application. Background Art

[0002] Gramseria parasuis It is a common opportunistic pathogen of the upper respiratory tract of pigs. Under certain conditions, it can cause pigs to suffer from arthritis, pericarditis, sepsis, pneumonia, pleurisy, emaciation and even acute death.

[0003] There are 15 serotypes of Gramseria parasuis, and some strains are untypeable, resulting in a lack of effective cross-protection between different serotypes. The main serotypes prevalent in my country are serotypes 4, 5, and 13 (Du Juan, Liu Yahui, et al., 2023). In recent years, due to the irrational use of antimicrobial drugs, drug resistance in Gramseria parasuis has continued to increase (Chen Xinhu, 2023). The use of non-antibiotic methods to prevent and control Gramseria parasuis will be an inevitable trend in the future. Vaccination is an important way to effectively prevent and control the occurrence and spread of Gramseria parasuis, reduce meat product contamination, and ensure food safety.

[0004] The traditional whole-cell inactivated vaccines currently widely used in clinical practice are serotype-specific and can only provide partial protection against the pathogen. They have the disadvantage of poor cross-protection against strains of different serotypes or even strains of the same serotype. Most traditional whole-cell inactivated vaccines mainly target serotypes 4 and 5 and cannot provide protection against serotype 13 strains. Subunit vaccines are safer but have problems such as insufficient immunogenicity. Natural attenuated vaccines have the risk of reversion to virulence.

[0005] As the main component of the outer membrane of Gram-negative bacteria, outer membrane proteins not only play an important role in maintaining the normal life activities of bacteria, but also have strong immunogenicity and cross-immunogenicity, and can serve as potential immune protective antigens.

[0006] Reverse vaccinology and immunoinformatics are rapidly developing and have proven to be excellent strategies for rapid vaccine prediction. In this study, we extracted outer membrane proteins from three clinical isolates of Gram's parasuis (serotype 4 HB04, serotype 5 SJZ05, and serotype 13 GD20) and a strain with reported good immunogenicity, MD0322. We performed 4D-label-free proteomics analysis to identify common outer membrane proteins among the four strains. We then used bioinformatics to screen for antigenic proteins. These identified proteins were further analyzed to predict B cell linear epitopes, helper T cell epitopes, and cytotoxic T cell epitopes. Dominant epitopes that were non-sensitizing, non-toxic, and highly antigenic were selected and concatenated using different linkers to create a final multi-epitope vaccine construct. This construct was then expressed in an Escherichia coli vector. Finally, the potential of the multi-epitope vaccine was preliminarily validated in a piglet challenge protection trial. Summary of the Invention

[0007] The object of the present invention is to provide a multi-epitope fusion protein targeting Gramseria parasuis antigens of multiple serotypes, wherein the fusion protein is shown in SEQ ID NO.2.

[0008] Another object of the present invention is to provide the use of the fusion protein in the preparation of Gramseria parasuis vaccine.

[0009] In order to achieve the above object, the present invention adopts the following technical measures:

[0010] The applicants conducted proteomic analysis of the outer membrane proteins of three clinically isolated strains HB04, SJZ05, and GD20 with high virulence and good immunogenicity, and the outer membrane protein of the strain MD0322 with high virulence and immunogenicity. Furthermore, the applicants further analyzed the eight antigenic proteins screened, predicted their B cell linear epitopes, helper T cell epitopes, and cytotoxic T cell epitopes, selected dominant epitopes that were non-sensitizing, non-toxic, and highly antigenic, and concatenated them through different linkers to obtain an immunogenic multi-epitope fusion protein that can recognize three serotypes. The fusion protein is shown in SEQ ID NO. 2.

[0011] The protection scope of the present invention includes:

[0012] A gene encoding the protein shown in SEQ ID NO.2;

[0013] The gene described above is preferably represented by SEQ ID NO.5.

[0014] An expression vector containing the above gene.

[0015] A recombinant microorganism expressing the protein shown in SEQ ID NO.2.

[0016] Application of the above substances in the preparation of Gramseria parasuis vaccine.

[0017] Application of the above substances in preparing medicines for treating or preventing Gramseria parasuis infection.

[0018] In the above application, preferably, the Gramseria parasuis is serotype 4, serotype 5 or serotype 13. Compared with the prior art, the present invention has the following advantages:

[0019] The present invention is the first to develop a multi-epitope fusion protein targeting multiple serotypes of Gramseria parasuis antigens. The fusion protein can be used as a Gramseria parasuis vaccine, and the protection rate against piglets infected with clinically isolated serotype 4 strain HB04, serotype 5 strain SJZ05 and serotype 13 strain GD20 reaches 100%, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A strategy for the tandem construction of multi-epitope fusion proteins.

[0021] Figure 2 It is used to detect specific antibodies against the multi-epitope fusion protein of Gramseria parasuis antigen.

[0022] Figure 3 Schematic diagram of the survival of Group A and Group D after being challenged with Gramseria parasuis serotype 4 clinical isolate HB04.

[0023] Figure 4 This is a schematic diagram of the survival status of Group B and Group E after being challenged with Gram-negative bacteria SJZ05, a clinical isolate of Gram-negative bacteria serotype 5.

[0024] Figure 5 Schematic diagram of the survival status of Group C and Group F after being challenged with Gramseria parasuis serotype 13 clinical isolate GD20.

[0025] Figure 6 These are typical autopsy lesions of Gramseria parasuis in groups A to F after infection. DETAILED DESCRIPTION

[0026] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. Unless otherwise specified, the experimental methods used in the examples of the present invention are conventional methods. The materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

[0027] Example 1:

[0028] Obtaining different serotypes of Gramseria parasuis and screening of antigenic epitopes:

[0029] 1. Gram-negative bacteria strains

[0030] Samples were collected from affected pig farms in 15 provinces and municipalities, including Hubei, Hunan, Hebei, and Guangdong. Affected pigs exhibited coughing, dyspnea, emaciation, lameness, and matted fur. Necropsy findings revealed pleurisy, pericarditis, peritonitis, arthritis, and meningitis. Gramseria parasuis was isolated from the samples, and strains of serotypes 4, 5, and 13 with good growth and reproduction rates were selected for pathogenicity and immunogenicity testing in piglets. The strains identified as serotype 4 HB04, serotype 5 SJZ05, and serotype 13 GD20 exhibited strong pathogenicity and immunogenicity.

[0031] 2. Extraction of Outer Membrane Proteins from Clinically Isolated Strains and Selection of Target Proteins

[0032] Outer membrane proteins (Beijing Biolab HR0095) were extracted from three clinical isolates, HB04, SJZ05, and GD20. The strain MD0322, reported to have high virulence and immunogenicity (Cai Xuwang, 2006), was also used as a control. These proteins were then submitted to Zhongke Xinsheng Biotechnology for 4D-label-free proteomics analysis to identify common outer membrane proteins among the four clinical isolates. The four clinical isolates shared 24 outer membrane proteins, which were further screened. Further screening was conducted based on physicochemical properties such as outer membrane antigenicity, theoretical isoelectric point, instability coefficient, fat coefficient, and overall average hydrophilicity. Finally, a comprehensive consideration of stable proteins with high antigenicity and hydrophilicity was selected as antigenic proteins. A total of eight antigenic proteins were obtained, as shown in Table 1 below.

[0033] Table 1 Screening results of 8 antigen proteins

[0034]

[0035] 3. Screening of protein epitopes

[0036] The dominant epitopes of 8 antigen proteins were predicted using servers such as BepiPred, IEDB, NetCTL-1.2 and NetMHCII 2.3. They were divided into linear B cell epitopes, toxic T cell epitopes and helper T cell epitopes. The antigenicity, sensitization and toxicity of the screened epitopes were predicted and analyzed, and epitopes with high antigenicity, non-sensitization and non-toxicity were selected as candidate dominant epitopes.

[0037] (1) The results of the B cell linear epitope screening of 8 antigenic proteins are shown in Table 2 below.

[0038] Table 2 Prediction results of B cell linear epitopes of 8 antigenic proteins

[0039]

[0040] (2) The results of the screening of helper T cell epitopes of 8 antigen proteins are shown in Table 3 below.

[0041] Table 3 Prediction results of helper T cell epitopes of 8 antigen proteins

[0042]

[0043]

[0044] (3) The results of screening for toxic T cell epitopes of 8 antigenic proteins are shown in Table 4 below.

[0045] Table 4 Prediction results of toxic T cell epitopes of 8 antigen proteins

[0046]

[0047] Example 2:

[0048] Tandem construction of multi-epitope fusion proteins:

[0049] 1. Multi-epitope fusion proteins in different tandem configurations:

[0050] The candidate B cell linear epitopes, helper T cell epitopes, and cytotoxic T cell epitopes selected from the eight antigenic proteins were combined in series to construct a multi-epitope fusion protein. Because the eight epitopes can be combined in different ways, the applicants conducted numerous experiments to determine the order of their connection. They found that the fusion proteins presented various issues, including expression stability, activity, and protective efficacy as vaccines. Ultimately, combination 2 was selected as the superior multi-epitope fusion protein for further animal testing.

[0051] The applicant provides the following three tandem construction strategies and protein expression results for illustration:

[0052] The candidate dominant epitopes were combined in different numbers and connection orders, and GPGPG, AAY and KK were used as linkers to connect helper T cell epitopes, cytotoxic T cell epitopes and linear B cell epitopes ( Figure 1 In combination 1 and combination 3, TLR2 agonist (Swiss-Prot DataBase accession number: A9JX08) was selected as a molecular adjuvant (Zhang Pengfei, 2022) and connected to the first helper T cell through the EAAAK linker ( Figure 1A) in order to optimize and screen the optimal construction strategy for recombinant proteins. The sequence of multi-epitope fusion protein combination 1 (YY1) is shown in SEQ ID NO. 1, the sequence of combination 2 (YY2) is shown in SEQ ID NO. 2, and the sequence of combination 3 (ZZ1) is shown in SEQ ID NO. 3.

[0053] 2. Construction and induced expression of different multi-epitope fusion proteins

[0054] The epitope fusion proteins constructed in tandem were codon-optimized for the three epitope protein coding gene sequences using a prokaryotic expression system (the optimized genes encoding combinations 1-3 are shown in SEQ ID NOs. 4-6). The synthesized optimized gene fragments were ligated into the pET-22b or pcold-TF prokaryotic expression vectors using the restriction endonucleases BamHI-XhoI or NdeI-HindIII, respectively, to construct recombinant plasmids. The polynucleotides for combinations 1 and 3 were ligated into pET-22b, and the polynucleotide corresponding to combination 2 was ligated into pcold-TF. The correctly identified recombinant plasmids were transformed into E.coil BL21 (DE3) host cells and, after resistance screening and recombinant plasmid identification, were used as engineered strains for expressing the recombinant proteins.

[0055] The optimal induction conditions were determined for each engineered strain according to the induction temperature, induction time, and inducer (IPTG) concentration. Finally, the engineered bacteria were transferred to 500 ml of antibiotic-containing culture medium at a volume ratio of 1:100 and shaken at 37°C and 200 rpm until the OD 600 When the p-value was between 0.5 and 0.7, IPTG was added to a final concentration of 0.5 mM, and pET-22b-YY1 and pET-22b-ZZ1 were induced at 37°C for 5 hours. For pCold-TF-YY2, the culture medium was pre-cooled to 16°C before adding the inducer, and then 0.5 mM IPTG was added and induced at 16°C for 24 hours. After induction, the cells were collected, crushed under high pressure, and centrifuged at low temperature. The supernatant precipitate was separated and subjected to SDS-PAGE electrophoresis. It was found that all three recombinant proteins were expressed in a soluble form, but the expression level of combination 1 (YY1) protein was low, and the expression level of combination 3 (ZZ1) protein was unstable from small to large amounts. Further exploration of the induction expression conditions is required in subsequent studies. Combination 2 (YY2) can be stably expressed in a soluble form. Therefore, combination 2 (YY2) is used as a candidate protein for the multi-epitope vaccine fused with the antigenic protein of Gramseria parasuis of the present invention.

[0056] The supernatant after bacterial cell disruption was collected, filtered through a 0.45 μm filter, and added to a pH-balanced affinity chromatography nickel column. The sample was repeated 3 to 4 times to allow the target protein to fully bind to the nickel column and then the flow-through was discarded. According to the binding of the protein to the nickel filler, a washing solution containing different concentrations of imidazole (20 to 50 mM) was used to remove impurities. Finally, the target protein was eluted with an eluent containing 500 mM imidazole. The target protein was verified by SDS-PAGE electrophoresis. Finally, the imidazole was removed by ultrafiltration and the buffer was replaced. Finally, 25 mg of YY2 protein was harvested from 500 ml of culture medium. The YY2 protein was aliquoted and stored at -80°C.

[0057] Example 3:

[0058] Application of Gramseria parasuis antigen multi-epitope fusion protein YY2 as a vaccine:

[0059] Reagents used in Groups A to C (reagent 1, experimental group): The antigen protein fused to the multi-epitope protein in Example 2 was quantified using a BCA protein quantification kit, the fusion protein concentration was adjusted, and the multi-epitope fusion protein YY2 was mixed with the water-in-oil-in-water formulation ISA 201VG adjuvant in a mass ratio of 1:1 to a protein concentration of 100 μg / ml. After emulsification, it was stored at 2 to 8°C for later use.

[0060] Reagents used in Groups D to F (reagent 2, control group): Sterile 1× PBS buffer (pH 7.4) and water-in-oil-in-water formulation ISA 201VG adjuvant were mixed at a mass ratio of 1:1, emulsified, and stored at 2-8°C until use.

[0061] 21-day-old newborn weaned piglets were randomly divided into experimental groups (groups A to C) and negative control groups (groups D to F). The experimental groups A to C were vaccinated with fusion protein multi-epitope vaccine 2 ml / head (reagent 1), and the control groups D to F were vaccinated with reagent 2 2 ml / head. The immunization method was intramuscular injection of the neck back. The second immunization was performed 21 days after the first immunization. 14 days after the second immunization, the clinical isolates of Gram-negative bacteria in Example 1 were used. Serum type 4 HB04, serum type 5 SJZ05 and serum type 13 GD20 strains were used to challenge the experimental groups A to C and the control groups D to F (i.e., the challenge strains of groups A and D were serum type 4 HB04, the challenge strains of groups B and E were serum type 5 SJZ05, and the challenge strains of groups C and F were serum type 13 GD20). The challenge doses of HB04, SJZ05 and GD20 were 1.2 × 10 10 CFU / head, 7.5×10 9 CFU / head and 1.5×10 10CFU / head, challenged by intrathoracic injection. Before immunization, 21 days after the first vaccination, and 14 days after the second vaccination, blood was collected from the anterior vena cava of the experimental and control pigs, and the serum was separated and stored for later use.

[0062] (1) Detection of serum specific antibody levels

[0063] The multi-epitope fusion protein YY2 was diluted to 200 ng / mL with PBS and coated on the ELISA plate, 100 μL per well, and coated at 4°C overnight. After coating, 5% BSA was used as a blocking agent and blocked at 37°C for 2 hours. The serum collected before immunization, 21 days after the first immunization, and 14 days after the second immunization (1:200) was used as the primary antibody, and goat anti-pig IgG-HRP (1:10000) was used as the secondary antibody. After sufficient incubation, TMB color development solution was used for 10 minutes, and the color development was stopped with color development stop solution, and then the OD was read using a microplate reader. 630 The numerical value of the fusion multi-epitope protein was detected and the difference in the specific antibody level of the two groups of serum was compared. Figure 2 . The specific antibody level of the experimental group was higher than that of the control group 21 days after the first vaccination; the specific antibody level of the experimental group was significantly higher than that of the negative control group 14 days after the second vaccination.

[0064] (2) Challenge protection test

[0065] The experimental groups (Groups A–C) and the negative control group (Groups D–E) were challenged 14 days after the second vaccination. Pigs were observed for 7 consecutive days after challenge, and clinical symptoms, incidence, and mortality were recorded in each group. After the observation period, surviving animals underwent pathological examinations, and pathological changes were recorded. The morbidity and mortality rates of each group were determined, ultimately determining the protective efficacy of the multi-epitope fusion vaccine containing G. parasuis antigenic proteins.

[0066] The experimental results showed that during the entire observation period, the pigs in the control group (groups D to F) developed symptoms such as difficulty breathing, loss of appetite, paddling, lameness, and hind limb paralysis; one pig in group D died on the first day of the observation period, and two pigs died on the second day; all pigs in group E died on the third day of the observation period; one pig in group F died on the first day of the observation period, two pigs died on the second day, and one pig died on the fourth day. Figures 3-5 Autopsies of sick and dead pigs revealed yellow effusions in the chest and abdominal cavities, adhesions between the chest and lungs, large amounts of fibrin exudates, and fibrinous exudates in the joints. Figure 6 During the entire observation period, no sick or dead pigs appeared in the experimental groups (Groups A to C). Autopsy of the experimental pigs showed that the chest cavity, abdominal cavity, joint cavity and lungs of the experimental pigs were normal ( Figure 6), indicating that the multi-serotype Gram-negative fusion protein vaccine, targeting multiple serotypes of Gram-negative serotypes, achieved a 100% protection rate against Gram-negative serotypes HB04, SJZ05, and GD20 in pigs infected with Gram-negative serotypes HB04, SJZ05, and GD20. Based on these test results, the vaccine effectively protects piglets against Gram-negative serotypes 4, 5, and 13. Specific test results are shown in Table 5 below.

[0067] Table 5 Protection rate of Gram-negative bacteria antigen protein fusion multi-epitope vaccine against clinical isolates after challenge

[0068]

Claims

1. An artificially synthesized protein, wherein the protein is represented by SEQ ID NO.

2.

2. A gene encoding the protein shown in SEQ ID NO.

2.

3. The gene according to claim 2, represented by SEQ ID NO.

5.

4. An expression vector containing the gene according to claim 2.

5. A recombinant microorganism expressing the protein shown in SEQ ID NO.

2.

6. Use of the protein according to claim 1, the gene according to claim 2, the expression vector according to claim 4 or the recombinant microorganism according to claim 5 in the preparation of a Gramseria parasuis vaccine, wherein the Gramseria parasuis is serotype 4, serotype 5 or serotype 13.

7. Use of the protein according to claim 1, the gene according to claim 2, the expression vector according to claim 4 or the recombinant microorganism according to claim 5 in the preparation of a medicament for preventing infection by Gramseria parasuis, wherein the Gramseria parasuis is serotype 4, serotype 5 or serotype 13.

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