Application of YnjE 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 YnjE protein in preparation of a clostridium perfringens resisting product. The amino acid sequence of the YnjE protein is as shown in SEQ ID NO. 1. The YnjE protein shows a high protection rate of 85.7% and high immunogenicity in immune protection in clostridium perfringens, in addition, as a surface exposed protein coded by a core gene, the YnjE protein is highly conserved in various clostridium perfringens, and due to the characteristic that the YnjE protein is easily recognized by an immune system, the YnjE protein can stimulate effective immune response, so that the immunogenicity of the YnjE protein is improved, and the immunogenicity of the YnjE protein is improved. The polypeptide has the potential of becoming a vaccine candidate.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology and biotechnology, and specifically relates to application of YnjE protein in preparing an anti-Clostridium perfringens product. Background Art

[0002] YnjE rhodamine-like domain proteins belong to an ancient and multifunctional protein superfamily, which has diverse physiological functions, including hydrolase activity, signal transduction, selenium metabolism, etc.

[0003] Clostrididium perfringens type A is the main pathogen causing hemorrhagic necrotic enteritis in cattle. At present, the specific pathogenic mechanism of hemorrhagic necrotic enteritis is not completely clear, but many studies have begun to explore its possible pathogenic pathways and try to screen out antigens with potential protective effects, such as α toxin (Plc), θ toxin (PFO), EF, FBA, Cyc and PNP proteins. At present, the research on the antigenic proteins of Clostridium perfringens is mostly focused on these toxin proteins, while the research on non-toxin proteins is relatively small. As a non-toxic protein, YnjE rhodamine-like domain protein needs further research on its application in order to further expand its scope of application. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the use of YnjE protein in the preparation of anti-Clostridium perfringens products. The YnjE protein has a high protection rate and high antibody titer. The protection rate of the YnjE protein after three immunizations reaches 85.7%.

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

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

[0007] The present invention has found in the study that the YnjE protein stimulates the body to produce a higher antibody titer. A lethal dose of ATCC13124 standard strain bacterial solution was intraperitoneally injected into mice, and the protection rate of the YnjE protein after three immunizations reached 85.7%. In addition, the YnjE protein is a surface-exposed protein encoded by a core gene. It is highly conserved in a variety of Clostridium perfringens, and because of its easy recognition by the immune system, it is more likely to stimulate an effective immune response and has the potential to become a vaccine candidate.

[0008] The second aspect of the present invention provides a recombinant expression vector comprising the gene encoding the YnjE protein.

[0009] In another preferred embodiment, the recombinant expression vector is obtained by inserting the coding gene of the YnjE protein between the BamHI and Xho I sites of the pET-32a plasmid;

[0010] The nucleotide sequence of the gene encoding the YnjE protein is shown in SEQ ID NO.2.

[0011] 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.

[0012] The fourth aspect of the present invention provides a polyclonal antibody, which is prepared using the YnjE protein or the recombinant engineered bacteria.

[0013] The fifth aspect of the present invention provides the use of the polyclonal antibody in the preparation of an anti-Clostridium perfringens product.

[0014] The sixth aspect of the present invention provides a vaccine comprising any one of the YnjE protein or the polyclonal antibody.

[0015] In another preferred embodiment, the vaccine further comprises a pharmaceutically acceptable adjuvant.

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

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

[0018] The YnjE protein of the present invention exhibits a high protection rate of 85.7% and high immunogenicity in Clostridium perfringens. In addition, as a surface-exposed protein encoded by a core gene, it is highly conserved in multiple Clostridium perfringens and is more likely to stimulate an effective immune response due to its easy recognition by the immune system, and has the potential to become a vaccine candidate.

[0019] The YnjE protein of the present invention has a high protection rate and high antibody titer, with a protection rate of 85.7%. The antibody titer after the third immunization exceeds 1:819200, and it has great potential for the development of new Clostridium vaccines. This protein is a surface-exposed protein encoded by the core gene. The core gene encoding means that the protein is highly conserved. Sequence comparison shows that the protein has a high coverage in A, B, C, D, E, F and G types of Clostridium perfringens, which shows that the protein is highly conserved in various types of Clostridium perfringens. Compared with proteins in the bacteria, surface-exposed proteins can be taken up and processed by APC cells and presented to T cells, and can also be directly recognized by the BCR receptors of B cells, that is, it is easier to stimulate the body to produce an immune response. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0023] Figure 4 The antibody titer diagram of three immunizations with YnjE protein; A is the antibody titer diagram of one immunization, B is the antibody titer diagram of two immunizations, and C is the antibody titer diagram of three immunizations.

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

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

[0026] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but 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 YnjE protein, and the research process is as follows: Figure 1 The amino acid sequence of the YnjE protein is shown in SEQ ID NO.1:

[0028]

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

[0030] MSLLKKLTLGLMAVSTTVTFMACSGNKSNETKTSIKSLSTKEVQENLKDNSWIAVDTRLSDSYNGWILYDEARGGHIPNAEDFSANWIKVDAKDKENKLDEALKTKGITTDKN IILYDANGEDSKVVADYLSKKGYKNLYSYDIKEWASDKDLPLEKYEDYSLIVPAKILKGVIDGNKPETFENAKNIKVLEASWGDENQTYGKGHVPTSVHINTDTIEPPPTWML ADDKTLTQFASDYGINKDDTIILTGAEQMAAYRLASVLKYMGVKDVRVLNGGDGAWTSAGYELETKVNPKTPGGDFGATIPADPEVITTIPELKEDLKKSDFTLVDNRTWDEY IGKISGYSYHDKKGRIEEGAVFGYAGENGPTSLEYYRNVDNTMRNGYEIEKMWEDQGINLKNHLVFMCGSGWRAAEVYTYANVMGLKNTSIYSDGWIGWSNGGNPSITGDPTK.

[0031] Example 1: Screening of YnjE protein

[0032] The YnjE protein was screened from the whole genome and proteome sequence information of 15 strains of bovine 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 included VaxiJen v2.0, IEDB, DiscoTope 3.0 and Epitope1D; pan-genome analysis used IPGA; and virulence factor analysis used the VFDB database.

[0034] The specific analysis method is as follows: First, the proteins in the genomes of 15 strains of Clostridium capsulatum were divided into potential antigens and non-antigens using Vaxijen. Potential antigens were predicted and located using pSORTb v3.03, CELLO v2.5, Loctree3 and Gpos-mploc subcellular localization tools. Those with at least three prediction tools localizing as extracellular and cell wall were defined as PSEs, and those predicted as cytoplasmic and cell membrane were eliminated. Those with inconsistent subcellular localization prediction results were defined as non-consensus proteins. SignalP4.1, Secretome2.0 and TMHMM2.0 tools were used to predict signal peptides, secretory peptides and transmembrane structures for non-consensus proteins. Proteins with signal peptides but no transmembrane structures were marked as PSEs, and those with signal peptides but no transmembrane structures were eliminated. Pan-genomic analysis was used to identify core genes and variable genes. Epitope1D, IEDB and Discotope3.0 were used to predict B cell linear and conformational epitopes for PSEs corresponding to core genes. The 30 proteins with the highest scores for each prediction result were called high-scoring proteins, and the remaining proteins were called low-scoring proteins. The target protein YnjE protein was determined based on whether there were high-scoring proteins with consistent prediction results between the software.

[0035] Example 2: Analysis of antigenic epitopes

[0036] The analysis method of YnjE 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 / ); T cell linear epitopes were predicted using IEDB prediction (http: / / tools.iedb.org / CD4episcore / ). B cell conformational epitopes were evaluated using weighted scores, and the results are shown in Figure 2. Figure 2 shown. Figure 2 The regions of medium and high-scoring B and T cell linear epitopes are marked in green and blue inside the protein; the regions of high-scoring B cell conformational epitopes are marked in red on the protein surface; and the overlapping regions of linear epitopes and conformational epitopes are marked in purple.

[0038] Example 3: Construction of prokaryotic expression vector

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

[0041] Table 1 Specific primers

[0042]

[0043] Example 4: YnjE protein expression and purification

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

[0045] The BL21-32a-YnjE overexpression vector plasmid was inoculated into LB liquid medium and induced for 8 h by 1 mM IPTG. After repeated freezing and thawing in liquid nitrogen and ultrasonic disruption, the YnjE protein was expressed in the form of inclusion bodies. The YnjE protein was obtained after Ni column affinity chromatography, dialysis and concentration. Figure 3 As shown. Figure 3 As shown. Figure 3 It can be seen that the immune effect of PLC protein is not as good as that of YnjE protein. Figure 3 The B in Figure 3 A in the figure is the effect of antibody binding and color development after transfer. The antibody only specifically binds to the target protein ESBP protein, as shown in b1 and b2. Therefore, there are no other extra bands on B. After the pET-32a plasmid protein and YnjE in B were transferred to the membrane by SDS-PAGE electrophoresis, the anti-his-tagged mouse IgG was used as the primary antibody for Western Blot identification. Specific bands appeared at the expected positions of 25kDa and 77kDa, respectively. In order to make the bands clearer, the background color was adjusted to white, as shown in b1 and b2. From b1 and b2, it can be seen that the 25kDa and 77kDa bands are clear, there are no extra bands, and their sizes are consistent with the molecular weight of the protein, indicating the high sensitivity and specificity of the pET-32a plasmid protein and YnjE protein.

[0046] Example 5: Antibody titer and cytokines

[0047] Dilute YnjE protein to 10 μg / mL using carbonate buffer (pH = 9.5), add to 96-well plate, 100 μL / well, and coat overnight at 4°C; wash 3 times with PBST, add 5% skim milk powder, 200 μL / well, and block at 37°C for 2 hours; wash 3 times with PBST, add mouse serum (gradient dilution starting from 1:400), 100 μL / well, and incubate at 37°C for 1 hour; wash 3 times with PBST, add HRP-labeled goat anti-mouse IgG (1:4000 dilution), 100 μL / well, and incubate at 37°C for 1 hour; wash 3 times with PBST, add TMB single-component colorimetric solution, 100 μL / well, and react at room temperature for 10 minutes; add 1 mol / L sulfuric acid solution, 100 μL / well to terminate the reaction. Detect OD450 with an enzyme reader. When the P / N value is greater than 2.1, it is judged as positive, and the corresponding maximum dilution multiple is judged as the antibody titer. The results are as follows Figure 4 As shown; cytokines were detected using sandwich ELISA kits, and the results were as follows Figure 5 shown.

[0048] from Figure 4 The figure below shows the changes in the polyclonal antibodies in the mouse serum one week after each immunization with YnjE protein. The antibody levels in the serum of mice immunized with protein were detected by ELISA. The results showed that after the first immunization, the antibody titer of YnjE protein was 1:12800; the antibody titer of the second and third immunizations was 1:819200 and 1:1638400 respectively. The antibody titer of YnjE protein was high in all three immunizations.

[0049] from Figure 5 The difference in cytokine levels induced by YnjE protein after infection of mice can be seen in the figure. PBS was used as the control group and YnjE protein was used as the immune protein group. The levels of IL-2, IL-4, IL-10 and IFN-γ in the serum of mice were detected by indirect ELISA 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 IL-10 secreted by mice in the YnjE protein group was significantly increased.

[0050] Example 6: Immunoprotective experiment of YnjE 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, a PBS control group and a YnjE group, with 10 mice in each group. Each mouse in the control group was injected with 150uL PBS solution; each mouse in the YnjE group was injected with 150uLYnjE protein solution diluted with PBS, with a protein content of 100ug. 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 intraperitoneally injected. 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 in the figure. Figure 6 As shown. The weight loss of mice was the largest within 48 hours after the virus attack, and the weight gradually increased after 3 days. The PBS control group had the largest weight loss, while the weight loss of the YnjE protein group was significantly less than that of the control group. In the survival curve, the survival rates of mice in the PBS and YnjE groups were 30% and 90%, respectively. The immune protection rates of the protein immunization groups were 85.7%. Based on the results of weight change and survival rate, YnjE protein has a good immune protection effect.

[0052] The immune protection rate calculation formula is as follows:

[0053] 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%.

[0054] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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.

[0055] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

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

1.

2. A recombinant expression vector, characterized in that: It comprises the gene encoding the YnjE protein as claimed in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that: The YnjE protein coding gene was inserted between the BamHI and XhoI sites of the pET-32a plasmid; The nucleotide sequence of the gene encoding the YnjE 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 YnjE 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 a product against Clostridium perfringens.

7. A vaccine, characterized in that Comprising the YnjE 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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