Multi-epitope fusion protein aiming at multi-serotype laisseria parasuis antigen and application of multi-epitope fusion protein

Abstract

The invention belongs to the field of animal vaccines, and particularly relates to a multi-epitope fusion protein aiming at multi-serotype laisseria parasuis antigen and application. The applicant carries out proteomics analysis on four clinical isolated strains of serotypes 4, 5 and 13 of the parasuis leather serotype 4, 5 and 13 with high toxicity and good immunogenicity, and eight antigen proteins are screened out of the four clinical isolated strains of the serotypes 4, 5 and 13 of the parasuis leather serotype 4, 5 and 13. And further analyzing the screened eight antigen proteins to finally obtain the antigen multi-epitope fusion protein with immunogenicity capable of recognizing three serotypes. When the fusion protein is used as a vaccine, the protection rate of clinically separated piglets infected with a serum type 4 strain HB04, a serum type 5 strain SJZ05 and a serum type 13 strain GD20 reaches 100%.

Description

Technical Field

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

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

[0003] There are 15 serotypes of Gramseria parasuis and some strains are untypeable, and there is a lack of effective cross-protection between different serotypes. The main serotypes prevalent in my country are serotype 4, serotype 5, and serotype 13 (Du Juan and Liu Yahui, 2023). In recent years, due to the irrational use of antibiotics, the drug resistance of 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 future development. Among them, vaccination is an important way to effectively prevent and control the occurrence and spread of Gramseria parasuis, reduce the contamination of meat products, 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. In addition, 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] At present, reverse vaccinology and immunoinformatics technology is developing rapidly, and it has been proven that this technology is an excellent strategy for rapid vaccine prediction. In this study, we extracted outer membrane proteins from three clinical isolates of Gramseria parasuis (serotype 4 HB04, serotype 5 SJZ05, serotype 13 GD20) and a reported strain MD0322 with good immunogenicity, and performed 4D-label free proteomics analysis to find the common outer membrane proteins of the four strains. The antigenic proteins were screened by bioinformatics technology, and the screened antigenic proteins were further analyzed to predict their B cell linear epitopes, helper T cell epitopes and toxic T cell epitopes. The dominant epitopes that are non-sensitizing, non-toxic and highly antigenic were selected, and they were connected in series through different linkers to obtain the final multi-epitope vaccine structure, which was then constructed on an Escherichia coli expression vector. Finally, the application potential of the multi-epitope vaccine was preliminarily verified in the piglet challenge protection test. Summary of the invention

[0007] The object of the present invention is to provide a multi-epitope fusion protein targeting multiple serotypes of Gramseria parasuis antigens, 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 applicant conducted proteomic analysis on the outer membrane proteins of three clinically isolated strains HB04, SJZ05, and GD20 with high virulence and good immunogenicity, and the outer membrane proteins of the strain MD0322 with high virulence and immunogenicity. Furthermore, the applicant further analyzed the 8 antigenic proteins screened out, predicted their B cell linear epitopes, helper T cell epitopes, and toxic T cell epitopes, selected non-sensitizing, non-toxic, and highly antigenic dominant epitopes, and connected them in series through different linkers to obtain an antigen multi-epitope fusion protein with immunogenicity that can recognize three serotypes, and 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 shown as 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] The invention relates to the use of the above-mentioned substance in preparing medicine 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 produce 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 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 status of Group A and Group D after being challenged with Gramseria parasuis serotype 4 clinical isolate HB04.

[0023] Figure 4 Schematic diagram of the survival status of group B and group E after being challenged with the clinical isolate SJZ05 of Gramseria parasuis serotype 5.

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

[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. The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

[0027] Embodiment 1:

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

[0029] 1. Isolation of Gramseria parasuis strains

[0030] Diseased materials were collected from diseased pig farms in 15 provinces and cities including Hubei, Hunan, Hebei, and Guangdong. Diseased pigs showed coughing, dyspnea, emaciation, lameness, and rough back hair; autopsy lesions showed pleurisy, pericarditis, peritonitis, arthritis, meningitis, etc. Parasuis Gramseriae were isolated from the diseased materials, and serotype 4, 5, and serotype 13 strains with good growth and reproduction speed were selected for piglet pathogenicity and immunogenicity tests, and strains with strong pathogenicity and good immunogenicity were selected, including serotype 4 HB04, serotype 5 SJZ05, and serotype 13 GD20.

[0031] 2. Extraction of outer membrane proteins from clinical isolates and selection of target proteins

[0032] The outer membrane proteins of three clinically isolated strains HB04, SJZ05, and GD20 (Beijing Biolab HR0095) were extracted, and the strain MD0322 (Cai Xuwang, 2006) reported to have high toxicity and immunogenicity was used as a control. The strains were submitted to Zhongke Xinsheng for 4D-label free proteomics analysis to find the outer membrane proteins common to the four clinically isolated strains. As a result, the four clinically isolated strains had 24 outer membrane proteins in total, and the 24 outer membrane proteins were further screened. Through further screening of the outer membrane protein antigenicity, theoretical isoelectric point, instability coefficient, fat coefficient, and total average hydrophilicity and other physicochemical properties, a stable protein with high antigenicity and hydrophilicity was finally selected as the antigen protein after comprehensive consideration, and a total of 8 antigen proteins were obtained. The specific results are shown in Table 1 below.

[0033] Table 1 Results of screening 8 antigen proteins

[0034]

[0035] 3. Screening of protein epitopes

[0036] The dominant epitopes of 8 antigen proteins were predicted by 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 antigenic proteins

[0042]

[0043]

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

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

[0046]

[0047] Embodiment 2:

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

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

[0050] The candidate B cell linear epitopes, helper T cell epitopes and toxic T cell epitopes selected from the 8 antigenic proteins were combined in series to construct a multi-epitope fusion protein. Since the 8 epitopes have different ways of series connection, the applicant has made a lot of attempts at their connection order and found that the fusion protein after series connection has various problems such as expression stability, activity or protection as a vaccine. Finally, combination 2 was selected as the superior multi-epitope fusion protein for further animal experiments.

[0051] The applicant provides the following three tandem construction strategies and protein expression conditions 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, toxic 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 best construction strategy of recombinant protein. 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 protein constructed in series was codon optimized for the coding gene sequences of the three epitope proteins according to the prokaryotic expression system (the optimized coding genes of combination 1-3 are shown in SEQ ID NO.4-6), and the synthesized optimized gene fragments were connected to the pET-22b or pcold-TF prokaryotic expression vectors through restriction endonucleases BamHI-XhoI or NdeI-HindIII to construct recombinant plasmids, respectively. The polynucleotides of combination 1 and combination 3 were connected to pET-22b, and the polynucleotides corresponding to combination 2 were connected to pcold-TF. The correctly identified recombinant plasmids were transformed into E.coil BL21 (DE3) host cells, and used as engineering strains for expressing recombinant proteins after resistance screening and recombinant plasmid identification.

[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 pH value was 0.5-0.7, IPTG with a final concentration of 0.5 mM was added, and pET-22b-YY1 and pET-22b-ZZ1 were induced at 37°C for 5 hours; pCold-TF-YY2 was precooled to 16°C before adding the inducer, and then 0.5 mM IPTG was added and induced at 16°C for 24 hours. After the induction was completed, the bacteria were collected, crushed under high pressure and centrifuged at low temperature, the supernatant precipitate was separated, and SDS-PAGE electrophoresis was performed. It was found that all three recombinant proteins could be expressed in a soluble form, but the expression level of combination 1 (YY1) protein was low, and the protein of combination 3 (ZZ1) was unstable from small amount expression to large amount expression. In subsequent studies, the induction expression conditions need to be further explored. Combination 2 (YY2) can be stably expressed in a soluble form, so combination 2 (YY2) is used as a candidate protein for the fusion multi-epitope vaccine of the parasuis antigen protein of the present invention.

[0056] The supernatant after bacterial cell disruption was collected, filtered with a 0.45 μm filter, and added to a pH-balanced affinity chromatography nickel column. The sample was loaded 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 the impurity protein. 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] Embodiment 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, test group): The antigen protein fusion 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 make the protein concentration of 100 μg / ml. After emulsification, it was stored at 2 to 8°C for standby 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, and after emulsification, they were stored at 2 to 8°C for use.

[0061] The 21-day-old newborn weaned piglets were randomly divided into experimental groups (groups A-C) and negative control groups (groups D-F). The experimental groups A-C were vaccinated with fusion protein multi-epitope vaccine 2 ml / head (reagent 1), and the control groups D-F were vaccinated with reagent 2 2 ml / head. The immunization method was intramuscular injection in the back of the neck. The second immunization was performed 21 days after the first immunization. 14 days after the second immunization, the clinical isolates of Gramsheria parasuis in Example 1 were used to attack the experimental groups A-C and the control groups D-F (that is, the attack strains of group A and group D were serum type 4 HB04, the attack strains of group B and group E were serum type 5 SJZ05, and the attack strains of group C and group F were serum type 13 GD20). The attack 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, the virus challenge method was intrathoracic injection. Before immunization, 21 days after the first immunization, and 14 days after the second immunization, blood was collected from the anterior vena cava of the experimental group and the control group, 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, overnight at 4°C, and then blocked at 37°C for 2 h using 5% BSA as a blocking agent. 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-swine IgG-HRP (1:10000) was used as the secondary antibody. After sufficient incubation, TMB colorimetric solution was used for color development for 10 min, and the color development was stopped with colorimetric stop solution, and then the OD was read using an enzyme reader. 630 The numerical value of the fusion multi-epitope protein was detected and compared between the two groups of serum specific antibody levels. 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 group (group A-C) and the negative control group (group D-E) were challenged 14 days after the second vaccination. The pigs in each group were observed for 7 consecutive days after the challenge, and the clinical symptoms, number of cases and number of deaths were recorded. After the observation period, the animals that did not die were pathologically dissected and their pathological changes were recorded. The morbidity and mortality of each group were determined, and the protection rate of the multi-epitope vaccine fused with the antigen protein of Gram-negative bacteria was finally determined.

[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 dyspnea, 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 to 5 ). Autopsies of sick and dead pigs revealed yellow effusion in the chest and abdominal cavities, adhesions between the chest and lungs, large amounts of cellulose exudates, and cellulose exudates in the joints. ( Figure 6 During the entire observation period, no sick pigs or dead pigs appeared in the test groups (groups A to C). The autopsy of the test pigs showed that the chest cavity, abdominal cavity, joint cavity and lungs of the test pigs were normal ( Figure 6), indicating that the multi-epitope fusion protein vaccine for multiple serotypes of Gramseria parasuis antigens has a 100% protection rate against Gramseria parasuis clinical isolates HB04, SJZ05 and GD20 in experimental pigs. Based on the above test results, the vaccine has a good protective effect against Gramseria parasuis serotype 4, 5 and 13 infections in piglets. The 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 isolated strains after challenge

[0068]

Claims

1. An artificially synthesized protein, wherein the protein is shown in SEQ ID NO.

2.

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

2.

3. The gene according to claim 2, is shown as SEQ ID NO.

5.

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

5. A recombinant microorganism expressing the protein represented by 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.

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 treating or preventing Gramseria parasuis infection.

8. The use according to claim 7 or 8, wherein the Gramseria parasuis is serotype 4, serotype 5 or serotype 13.

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