Application of ESBP protein in preparation of anti-clostridium perfringens product

Abstract

The invention belongs to the technical field of molecular biology and biology, and particularly relates to application of ESBP protein in preparation of a clostridium perfringens resisting product. The amino acid sequence of the ESBP protein is as shown in SEQ ID NO. 1. The ESBP protein shows a 100% high protection rate and high immunogenicity in immune protection in clostridium perfringens, in addition, as a surface exposed protein coded by a core gene, the ESBP protein is highly conserved in various clostridium perfringens, and due to the characteristic that the ESBP protein is easy to recognize by an immune system, the ESBP protein is more likely to excite an effective immune response, so that the ESBP protein can be used as a surface exposed protein for immunoprotection of the clostridium perfringens. The polypeptide has the potential of becoming a vaccine candidate.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and biotechnology, and particularly relates to the application of ESBP protein in the preparation of an anti-Clostridium perfringens product. Background Art

[0002] Clostrididium perfringens type A is the primary pathogen causing hemorrhagic necrotic enteritis in cattle. The disease is easily disseminated among cattle, with rapid onset and high mortality. Therefore, there is an urgent need to identify proteins with strong immunogenicity and high protective rates for studying pathogenic mechanisms, developing new vaccines, and evaluating the immune efficacy of existing vaccines.

[0003] Although the specific pathogenic mechanism of hemorrhagic necrotizing enterocolitis remains incompletely understood, numerous studies have begun to explore its potential pathogenic pathways and attempt to identify antigens with potential protective effects, such as alpha toxin (Plc), theta toxin (PFO), EF, FBA, Cyc, and PNP proteins. Currently, research on the antigenic proteins of Clostridium perfringens has largely focused on these toxin proteins, while relatively little research has been conducted on non-toxin proteins. Despite this, evidence suggests that non-toxin proteins play a crucial role in Clostridium infection and may provide important protective effects. The ESBP protein is a non-toxin protein belonging to the extracellular solute-binding protein family, which plays an important role in infections with Streptococcus pneumoniae and Staphylococcus aureus. Therefore, analyzing the function of the ESBP protein and exploring its application in preventing Clostridium perfringens infection is of great significance. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides the use of ESBP protein in the preparation of anti-Clostridium perfringens products. ESBP protein stimulates the body to produce higher antibody titers, and the protection rate of ESBP protein after three immunizations reaches 100%.

[0005] To achieve the above objectives, the technical solutions of the present invention are specifically as follows.

[0006] In a first aspect, the present invention provides the use of ESBP protein in preparing an anti-Clostridium perfringens product. The amino acid sequence of the ESBP protein is shown in SEQ ID NO.1.

[0007] This study uses reverse vaccinology to identify surface-expressed proteins with high immune scores from proteins encoded by 2,304 core genes and 3,606 variable genes in 15 strains of C. perfringens that cause bovine pathogenicity. The study found that ESBP is a surface-exposed protein encoded by the core gene. This core gene encoding ESBP is highly conserved. Sequence alignment revealed high coverage and similarity among C. perfringens genotypes A, B, C, D, E, F, and G. This suggests that ESBP is highly conserved across all strains. Compared to proteins found within the bacterium, surface-exposed proteins can be taken up, processed, and presented to T cells by APCs and directly recognized by B cell receptors (BCRs), making them more likely to stimulate an immune response. Although ESBP proteins belong to the same protein family, they differ significantly. Sequence alignment results confirm this finding. The ESBP protein identified in this study is highly conserved only in C. perfringens; all proteins in this family from other strains share less than 75% similarity to the ESBP identified in this study. Therefore, the present invention screened out a new ESBP protein for the first time and verified its function.

[0008] The protein was expressed and purified, and mice were immunized to obtain polyclonal antibody serum. Antibody titers were measured by indirect ELISA, cytokine levels were measured to analyze the type of immune response, and protection against infection was calculated using challenge and protection experiments. The results showed that ESBP protein stimulated the production of high antibody titers. When mice were intraperitoneally injected with a lethal dose of the ATCC13124 standard strain, the protection rate of ESBP protein reached 100% after three immunizations.

[0009] The second aspect of the present invention provides a recombinant expression vector comprising the ESBP protein.

[0010] In another preferred embodiment, the recombinant expression vector is obtained by inserting the ESBP protein coding gene into the BamHI and XhoI sites of the pET-32a overexpression vector plasmid;

[0011] The nucleotide sequence of the gene encoding the ESBP protein is shown in SEQ ID NO.2.

[0012] The third aspect of the present invention provides a recombinant engineered bacterium comprising the recombinant expression vector, wherein the recombinant engineered bacterium is obtained by transferring the recombinant expression vector into competent cells E. coli DH5α.

[0013] The fourth aspect of the present invention provides a polyclonal antibody, which comprises any one of the ESBP protein, the recombinant expression vector, and the recombinant engineered bacteria.

[0014] The fifth aspect of the present invention provides the use of the polyclonal antibody in the preparation of an anti-Clostridium perfringens product.

[0015] The sixth aspect of the present invention provides a vaccine comprising any one of the ESBP protein, the recombinant expression vector, the recombinant engineered bacteria, the polyclonal antibody, and a pharmaceutically acceptable adjuvant.

[0016] The seventh aspect of the present invention provides the use of the vaccine in inhibiting Clostridium perfringens.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The ESBP protein of the present invention exhibits a high protection rate of 100% and high immunogenicity in immune protection against Clostridium perfringens. In addition, as a surface-exposed protein encoded by a core gene, it is highly conserved in multiple Clostridium perfringens strains and can stimulate an effective immune response due to its easy recognition by the immune system. The ESBP protein provides a new approach for exploring the pathogenic mechanism of Clostridium perfringens.

[0019] (2) The ESBP protein of the present invention has a high protection rate and high antibody titer, with a protection rate of 100% and an antibody titer exceeding 1:819200 after the third immunization, demonstrating the ability to prepare a new Clostridium vaccine. The ESBP protein is deeply involved in the bacterial uptake of peptides, metal ions, and carbohydrates from the environment, and the uptake of metal ions may be highly correlated with its virulence. This provides a theoretical basis for further research into the function of this protein and will contribute to the exploration of the pathogenic mechanism of Clostridium perfringens type A. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Diagram of the research and analysis process of ESBP protein.

[0021] Figure 2 This is the antigen epitope distribution map of ESBP protein.

[0022] Figure 3 The expression and purification diagrams of ESBP protein; A is the SDS-PAGE detection result of protein expression, B is the WB verification result of protein expression, and C is the ESBP protein purification result. M in the figure represents protein marker, 0 represents pET-32a plasmid protein, 1 represents PLC protein, and 2 represents pET-32a-ESBP protein; 2a represents the purified ESBP protein; b1 in B is the sample diagram, and b2 is the white diagram.

[0023] Figure 4These are antibody titer diagrams for three immunizations with ESBP protein, where A is the antibody titer diagram for one immunization, B is the antibody titer diagram for two immunizations, and C is the antibody titer diagram for three immunizations.

[0024] Figure 5 This is a diagram showing the changes in cytokines after immunization with protein.

[0025] Figure 6 These are the result graphs of the weight change rate and survival rate of mice, where A is the result graph of the weight change of mice, and B is the result graph of the survival rate of mice. DETAILED DESCRIPTION

[0026] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0027] The present invention provides a new use of ESBP protein, and its specific research process is as follows: Figure 1 The amino acid sequence of the ESBP protein is shown in SEQ ID NO.1:

[0028] MRKKILSAILCMIGATAMVGCGDKTDSTQAKDGGKVKLRMTNWNNEDTMKDMLKYLSEKLPNVEIEYQFIDNSNYNTIVDTQLSAEEGPDIICESPASALKHAK LGYLENVNDLAKKYSDSGTNVYKYDNNVYALPGISWFEGIYYNEKLFEENNIQIPKTFDEYIEVCKKFQSLGIKPLAAGLKSWEPLLKNSMAFVTAEYLSTDAGK NFGQEYREGKVKLDGTWNMYLDKWSEMIKDGIYTKDMTGIDHDQALEEFATGKAAMYCSGPWDLEAIMSKNPDLKLNMMPFYGTKPSDGWLIGGPGCGFAVNSKS KNKDAAMEVLKAISTEEGQKALWENNQGGSSYLTGTSFTLPEAFKGAEKAINAGHIYCPWNEWGDAGSAHVDYGKQMQNYLLGNQDLKTTLSNVDSAASELINK.

[0029] The nucleotide sequence of the gene encoding the ESBP protein is shown in SEQ ID NO.2:

[0030]

[0031] Example 1: Screening of ESBP protein

[0032] The ESBP protein was screened from the whole genome and proteome sequence information of 15 strains of necrotizing hemorrhagic enteritis from China, the United States, and Argentina. The analysis tools are as follows:

[0033] Protein subcellular localization prediction tools used were pSORTb v3.03, CELLO v2.5, Loctree 3, and Gpos-mploc; immunogenicity prediction tools were VaxiJen v2.0, IEDB, DiscoTope 3.0, and Epitope1D; pan-genome analysis was performed using IPGA; and virulence factor analysis was performed using the VFDB database.

[0034] The specific analysis method is as follows: First, proteins from the genomes of 15 Clostridium perfringens strains were classified as potential antigens or non-antigens using Vaxijen. Potential antigens were localized using the pSORTb v3.03, CELLO v2.5, Loctree3, and Gpos-mploc subcellular localization tools. Proteins predicted as extracellular and cell wall by at least three of the three tools were defined as PSEs. Proteins predicted as cytoplasmic and cell membrane were excluded. Proteins with inconsistent subcellular localization predictions were defined as non-consensus proteins. Signal peptides, secretory peptides, and transmembrane structures were predicted for non-consensus proteins using SignalP4.1, Secretome2.0, and TMHMM2.0. Proteins with signal peptides but no transmembrane structure were labeled as PSEs, while those with negative predictions were excluded. Pan-genome analysis was used to identify core and variable genes. PSEs corresponding to core genes were predicted for B cell linear and conformational epitopes using Epitope1D, IEDB, and Discotope3.0. The 30 proteins with the highest scores for each prediction were designated high-scoring proteins, and the remaining proteins were designated low-scoring proteins. The target protein ESBP protein was determined based on whether there were consistent high-scoring proteins predicted by the software.

[0035] Example 2: Analysis of antigenic epitopes

[0036] The analysis method of ESBP protein antigen epitope is as follows:

[0037] B cell linear epitopes were predicted using IEDB (http: / / tools.iedb.org / bcell / ); B cell conformational epitopes were predicted using DiscoTope 3.0 (https: / / services.healthtech.dtu.dk / services / DiscoTope-3.0 / ); and T cell linear epitopes were predicted using IEDB (http: / / tools.iedb.org / CD4episcore / ). B cell conformational epitopes were evaluated using weighted scores, and the results are shown in Figure 2. Figure 2 As shown, Figure 2 Medium- and high-scoring B and T cell linear epitope regions are marked in green and blue inside the protein; high-scoring B cell conformational epitope regions are marked in red on the protein surface; and overlapping regions between linear and conformational epitopes are marked in purple.

[0038] Example 3: Construction of prokaryotic expression vector and recombinant engineering bacteria

[0039] Materials: ATCC13124 strain, specific primers as shown in Table 1, restriction enzymes BamHI and XhoI, vector pET-32a, competent cells E. coli DH5α and E. coli BL21 (DE3). The specific process is as follows:

[0040] The ESBP gene was amplified by PCR using the primers in Table 1. The PCR product was detected and recovered by 1% agarose gel electrophoresis. The ESBP gene and the pET-32a plasmid were then double-digested with the restriction endonucleases BamHI and EcoRI. The digested products were ligated using T4 ligase, and the ligated products were transformed into E. coli DH5α competent cells. After 12 hours at 37°C, a single colony was picked and cultured, and PCR verification was performed using the 32a verification primers. For successfully verified bacteria, the pET-32a-ESBP plasmid was extracted using a plasmid miniprep kit and sent to Xinjiang Youkang Biological Co., Ltd. for sequencing. The recombinant plasmid was transformed into the expression strain BL21 to construct the expression strain BL21-32a-ESBP.

[0041] Table 1 Specific primers

[0042]

[0043] Example 4: ESBP protein expression and purification

[0044] Materials: sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) preparation kit, isopropylthiogalactoside (IPTG) and Ni column packing.

[0045] The BL21-32a-ESBP overexpression vector plasmid was inoculated into LB liquid medium and induced with 1 mM IPTG for 8 h. After repeated freezing and thawing in liquid nitrogen and ultrasonic disruption, the ESBP protein was expressed in a soluble form. The ESBP protein was obtained after Ni column affinity chromatography, dialysis, and concentration. Figure 3 As shown. Figure 3 It can be seen that the immune effect of PLC protein is not as good as that of ESBP protein. Figure 3 B in the figure is Western blotting, which involves separating proteins by electrophoresis, transferring the proteins to a membrane, and then detecting the target protein ESBP protein using a specific antibody. Figure 3 The B in Figure 3 Figure A shows the effect of antibody-mediated staining after transfer to a membrane. The antibody specifically binds to the target protein, ESBP, resulting in the absence of any other extraneous bands in Figure B. In Figure B, pET-32a plasmid protein, PLC protein, and ESBP protein were transferred to a membrane by SDS-PAGE electrophoresis. Western blot analysis using anti-His-tagged mouse IgG as the primary antibody revealed specific bands at the expected positions of 25 kDa, 70 kDa, and 75 kDa, respectively. To enhance the clarity of the bands, the background color was adjusted to white, as shown in Figures b1 and b2. As can be seen from Figures b1 and b2, the 25 kDa, 70 kDa, and 75 kDa bands are clear, with no extraneous bands. Their sizes are consistent with the protein molecular weights, demonstrating high sensitivity and specificity for pET-32a plasmid protein, PLC protein, and ESBP protein.

[0046] Example 5: Antibody titer and cytokines

[0047] ESBP protein was diluted to 10 μg / mL using carbonate buffer (pH = 9.5) and added to a 96-well plate at 100 μL / well. The plate was coated overnight at 4°C. The plate was washed three times with PBST, and 5 wt% skim milk powder was added at 200 μL / well. The plate was blocked at 37°C for 2 h. The plate was washed three times with PBST, and mouse serum (1:400 dilution starting at 1:400) was added at 100 μL / well. The plate was incubated at 37°C for 1 h. The plate was washed three times with PBST, and HRP-labeled goat anti-mouse IgG (1:4000 dilution) was added at 100 μL / well. The plate was incubated at 37°C for 1 h. The plate was washed three times with PBST, and TMB single-component colorimetric solution was added at 100 μL / well. The reaction was allowed to react at room temperature for 10 min. The reaction was terminated by adding 1 mol / L sulfuric acid solution at 100 μL / well. The OD450 value was measured using a microplate reader. A P / N value greater than 2.1 was considered positive, and the corresponding maximum dilution factor was determined as the antibody titer. The results are shown in Figure 2. Figure 4 Cytokines were detected using sandwich ELISA kits, and the results were as shown in Figure 5 shown.

[0048] from Figure 4The figure below shows the changes in polyclonal antibodies in mouse serum one week after each immunization with ESBP protein. ELISA was used to measure antibody levels in the serum of mice immunized with the protein. The results showed that after the first immunization, the antibody titer for ESBP protein was 1:12,800; after the second and third immunizations, the antibody titer was 1:819,200 and 1:1,638,400, respectively. ESBP protein antibody titers were high across all three immunizations.

[0049] from Figure 5 The differences in cytokine levels induced by ESBP protein after infection of mice can be seen in the data. PBS buffer served as the blank control, while PLC protein, Ynje protein, and ESBP protein served as the immune protein groups. Indirect ELISA was used to measure IL-2, IL-4, IL-10, and IFN-γ levels in mouse serum one week after the second immunization. The results showed that compared with the immune protein group, the PBS control group secreted significantly more IL-2, IL-4, and IFN-γ (P < 0.05). The ESBP protein group also secreted significantly more IL-10. ESBP protein stimulated the release of IL-2, IL-4, IL-10, and IFN-γ in mouse serum, primarily inducing a Th2 immune response.

[0050] Example 6: Immunoprotective experiment of ESBP protein

[0051] Twenty SPF-grade 6-week-old BALB / c mice were purchased from Beijing Sibeifu Biotechnology Co., Ltd. and raised in the Animal Experimental Center of Shihezi University. The 20 female Balb / C mice were divided into two groups, namely the PBS control group and the ESBP group, with 10 mice in each group. Each mouse in the control group was injected with 150 μL PBS solution; each mouse in the ESBP group was injected with 150 μL ESBP protein solution diluted with PBS, with a protein content of 100 μg. The immunization method was intraperitoneal injection, with a total of three immunizations at intervals of 2 weeks. One week after the immunization, blood was collected to separate the serum. Ten days after the third immunization, a lethal dose of Clostridium perfringens PBS resuspension was injected intraperitoneally. The absorbance of the resuspension at OD600 was 0.9. At the same time, the weight changes of the mice within 7 days of the infection were recorded. The weight change rate (percentage) and immune protection rate of the mice were calculated, and the results are shown as follows. Figure 6As shown in the figure, the weight loss of mice was the largest within 48 hours after infection, and the weight gradually increased after 3 to 4 days. Among them, the PBS control group had the largest weight loss, and the ESBP group had the smallest weight loss. In the survival curve, the survival rates of mice in the PBS, Plc, ESBP and YnjE groups were 30%, 80%, 100% and 90%, respectively. The immune protection rates of the protein immunization groups were 71.4%, 100% and 85.7%, respectively. Based on the results of weight changes and survival rates, all three proteins have good immune protection effects, among which the protective effect of ESBP is more prominent. The formula for calculating the immune protection rate is as follows:

[0052] Immune protection rate (%) = (number of deaths of mice in the control group - number of deaths of mice in the immunized group) / number of deaths of mice in the control group × 100%.

[0053] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0054] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. The use of ESBP protein in the preparation of anti-Clostridium perfringens products, characterized in that: The amino acid sequence of the ESBP protein is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: It comprises the gene encoding the ESBP protein according to claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The ESBP protein coding gene is inserted into the BamHI and XhoI sites of the pET-32a overexpression vector plasmid to obtain the protein; The nucleotide sequence of the gene encoding the ESBP protein is shown in SEQ ID NO.

2.

4. A recombinant engineered bacterium according to claim 3, characterized in that: The recombinant expression vector is transferred into competent cells E.coli DH5α to obtain the protein.

5. A polyclonal antibody, characterized in that It is prepared using the ESBP protein described in claim 1 or the recombinant engineering bacteria described in claim 4.

6. Use of the polyclonal antibody according to claim 5 in the preparation of an anti-Clostridium perfringens product.

7. A vaccine, characterized in that It comprises the ESBP protein according to claim 1 or the polyclonal antibody according to claim 5.

8. The vaccine according to claim 7, characterized in that Pharmaceutically acceptable adjuvants are also included.

9. Use of the vaccine according to claim 7 in inhibiting Clostridium perfringens.

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