Application of SMP and AMD proteins in preparation of vaccine for resisting arcanobacterium pyogenes

Abstract

The invention relates to the technical field of biology, and discloses an application of SMP and AMD proteins in preparation of an anti-arcanobacterium pyogenes vaccine. The amino acid sequence of the SMP protein is as shown in SEQ ID NO. 1. The amino acid sequence of the AMD protein is as shown in SEQ ID NO. 2. The vaccine for resisting the Trupererella pyogenes comprises an SMP protein with an amino acid sequence as shown in SEQ ID NO.1 and / or an AMD protein with an amino acid sequence as shown in SEQ ID NO.2, and an adjuvant. According to the application of the SMP and AMD proteins in the preparation of the vaccine for resisting the arcanobacterium pyogenes, the prepared protein vaccine can stimulate a body to generate a large number of antibodies, resist T.pyogenes attack, relieve body damage and improve the survival rate, and has important application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of SMP and AMD proteins in the preparation of vaccines against Trueperella pyogenes. Background Art

[0002] Trueperella pyogenes (T. pyogenes), also known as Arcanobacterium pyogenes, is a Gram-positive, pleomorphic, non-motile, sporeless, facultative anaerobic microorganism. Reports of related cases have been found in pigs, cattle, sheep and other animals of important economic value. T. pyogenes is a conditional pathogen that usually colonizes parts such as the urogenital tract, respiratory tract, gastrointestinal mucosa and mammary glands of animals. When animals are stressed, suffer from primary diseases and mechanical injuries, resulting in impaired immune function, T. pyogenes can multiply in large numbers and invade deep into tissues, causing suppurative infections of the skin, organs and joints, leading to arthritis, mastitis, endocarditis, osteomyelitis, endometritis, liver abscess, spermatorrhea, miscarriage, stillbirth and other diseases, bringing serious economic losses to the livestock industry.

[0003] At present, infections caused by T. pyogenes can be prevented or treated with antibiotics. Some studies have shown that more than 50% of the isolated strains are resistant to amoxicillin, ampicillin, chloramphenicol, florfenicol, oxytetracycline and penicillin. Whole-genome sequencing of T. pyogenes isolates from various animals has found that common drug-resistant genes in Gram-negative bacteria exist in T. pyogenes strains from different animal sources, indicating that the bacterium has broad antibiotic resistance. Given the emergence of multi-drug resistant strains of T. pyogenes, the use of antibacterial drugs needs to be cautious. Therefore, in order to prevent and control the infection of this bacterium, new preparations for the prevention and control of T. pyogenes infection need to be developed.

[0004] Vaccines against T. pyogenes are of great significance for the prevention and control of the disease. Since the 1950s, different forms of T. pyogenes vaccines have been developed and evaluated. Researchers once inactivated the supernatant of T. pyogenes cultures with formaldehyde and adsorbed it with aluminum hydroxide gel to prepare inactivated vaccines. However, after immunization, animals could not obtain effective immune protection. In the previous work of the inventor, an aluminum hydroxide gel-adsorbed inactivated vaccine against T. pyogenes was also prepared and inoculated into mice. The research found that when the product of T. pyogenes was concentrated 4-fold, the inactivated vaccine could provide complete immune protection for mice. This shows that the antigen content in the T. pyogenes vaccine is directly related to the immune protection effect.

[0005] However, there are still many drawbacks in the production process of such vaccines. First, the growth cycle of T.pyogenes is relatively long and its nutritional requirements are high. Generally, animal serum is needed as a supplement to the culture medium to promote growth, increasing the production cost of the vaccine. Second, inactivated vaccines may contain microbial metabolites and other chemical impurities, which can affect the immune effect. Finally, vaccines prepared by this technology contain a large amount of animal-derived components, which may cause biosafety problems or side effects. For example, the serum components in the vaccine may transmit some infectious diseases, such as mad cow disease, or cause hypersensitivity reactions in animals. At present, the immune protection effect of commercially available inactivated T.pyogenes vaccines on the foreign market is not ideal.

[0006] With the continuous in-depth research on T.pyogenes vaccines, several immunogens that can provide immune protection for animals have been discovered. Some studies inactivated three bacteria (T.pyogenes, Escherichia coli, and Clostridium necrotizing) and mixed them with recombinantly expressed bacterial virulence factors (FimH, LKT, and PLO) to prepare vaccines with five different antigen combinations. The results showed that subcutaneous injection of the vaccine could significantly reduce the incidence of uterine infections during the puerperium of dairy cows, thus improving reproductive performance. Some studies concatenated the coding genes of PLO, CbpA, FimA, and NanH into a chimeric gene and linked a CpG ODN 1826 motif. Then the chimeric gene was ligated into an expression plasmid vector, encapsulated with chitosan nanoparticles to protect the DNA, and the chimeric DNA vaccine was inoculated into mice by intramuscular injection to generate antibodies against PLO, CbpA, NanH, and FimA.

[0007] Currently, the exploration of immunogens mainly focuses on the major virulence factors of bacteria. Although there have been some achievements, the results show that developing vaccines relying solely on these virulence factors cannot provide complete immune protection against T.pyogenes infection. Our team previously cloned the HtaA protein coding gene and mixed it with the PLO genetic engineering toxoid - PLO W497F for immunization, providing effective immune protection for mice. This further indicates that the exploration and utilization of potential immunogens should not be limited to known virulence factors, and many potential immunogens that have been discovered but not studied yet still have important research value. Summary of the Invention

[0008] The object of the present invention is to provide the application of SMP and AMD proteins in the preparation of vaccines against Arcanobacterium pyogenes. The prepared protein vaccine can help the body produce a large amount of antibodies, resist the attack of T.pyogenes, reduce body damage, and improve the survival rate, having important application value.

[0009] To achieve the above object, the present invention provides the use of SMP and AMD proteins in the preparation of a vaccine against Arcanobacterium pyogenes, which is applied to the preparation of a vaccine against T. pyogenes.

[0010] The present invention also provides the use of SMP protein in the preparation of a vaccine against T. pyogenes.

[0011] Furthermore, the amino acid sequence of the SMP protein is as shown in SEQ ID NO.1.

[0012] The present invention also provides the use of AMD protein in the preparation of a vaccine against T. pyogenes.

[0013] Furthermore, the amino acid sequence of the AMD protein is as shown in SEQ ID NO.2.

[0014] The present invention also provides a vaccine against T. pyogenes, which comprises the SMP protein with the amino acid sequence as shown in SEQ ID NO.1 and / or the AMD protein with the amino acid sequence as shown in SEQ ID NO.2 and an adjuvant.

[0015] Furthermore, the adjuvant is aluminum hydroxide.

[0016] The present invention also provides a drug for preventing T. pyogenes, which comprises the SMP protein with the amino acid sequence as shown in SEQ ID NO.1 and / or the AMD protein with the amino acid sequence as shown in SEQ ID NO.2.

[0017] The advantages and positive effects of the use of the SMP and AMD proteins in the preparation of a vaccine against T. pyogenes according to the present invention are as follows:

[0018] The present invention provides a vaccine against T. pyogenes, which includes the SMP protein and the AMD protein. This protein vaccine can stimulate the body to produce a large amount of antibodies, resist the attack of T. pyogenes, simultaneously regulate the expression of inflammatory cytokine genes, reduce histopathological lesions, and improve the survival rate, having important application value.

[0019] The technical solution of the present invention will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the animal immunization and experimental protocol in the embodiment of the present invention;

[0021] Figure 2 It is the expression and purification of the recombinant protein in the embodiment of the present invention;

[0022] Figure 3Results of the determination of specific antibody levels after immunization and bacterial challenge in the embodiments of the present invention, where A is the antibody titer of SMP and B is the antibody titer of AMD;

[0023] Figure 4 Skin damage and body weight recovery of mice after bacterial challenge in the embodiments of the present invention, where A is the wound recovery of each group of mice at different time periods after bacterial challenge, and the image J statistical wound superposition analysis diagram; B is the wound closure rate of the surviving mice in each group at different time points after bacterial challenge; C is the statistical trend of the body weight change of the surviving mice from before bacterial challenge to the 11th day after bacterial challenge. The data are expressed as SEM ± mean value, and one-way analysis of variance (ANOVA) is used, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001;

[0024] Figure 5 Results of histopathological observation in the embodiments of the present invention;

[0025] Figure 6 Detection of the relative expression levels of various inflammatory cytokines in serum and organs after bacterial challenge in the embodiments of the present invention, where A is the detection result of TNF-α level in each group of organs; B is the detection result of IL-1β level in each group of organs; C is the detection result of IL-6 level in each group of organs; D is the detection result of IL-8 level in each group of organs; E is the detection result of IL-10 level in each group of organs; F is the detection of IL-1β expression level in each group of sera; G is the detection of IL-10 expression level in each group of sera. The data are expressed as SEM ± mean value;

[0026] Figure 7 Evaluation results of bacterial clearance rate and survival rate protection effect in the embodiments of the present invention, where A is the antibody titer of PLO in each group; B is the survival rate in each group. The data are expressed as SEM ± mean value, and one-way analysis of variance (ANOVA) is used, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Detailed implementation manners

[0027] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0029] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are generally determined according to national standards. For the experimental instruments, equipment, and reagents without sources noted in the following embodiments, they are all commercially available raw materials.

[0030] Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the methods of the present invention.

[0031] The Trueperella pyogenes in the present invention is specifically Trueperella pyogenes (T. pyogenes strain 0912), which is recorded in the following literature: Liang H, Wang B, Wang J, Ma B, Zhang W. Pyolysin of Trueperella pyogenes Induces Pyroptosis and IL-1β Release in Murine Macrophages Through Potassium / NLRP3 / Caspase-1 / Gasdermin D Pathway. Front Immunol. 2022 Mar 15;13:832458. Hu Y., Zhang W., Bao J., Wu Y., Yan M., Xiao Y., Yang L., Zhang Y., Wang J. A chimeric protein composed of the binding domains of Clostridium perfringens phospholipase c and Trueperella pyogenes pyolysin induces partial immunoprotection in a mouse model. Res Vet Sci. 2016;107:106-115. Yang L, Liang H, Wang B, Ma B, Wang J, Zhang W. Evaluation of the Potency of Two Pyolysin-Derived Recombinant Proteins as Vaccine Candidates of Trueperella Pyogenes in a Mouse Model: Pyolysin Oligomerization and Structural Change Affect the Efficacy of Pyolysin-Based Vaccines. Vaccines (Basel). 2020 Feb 10;8(1):79. Trueperella pyogenes (T. pyogenes strain 0912) is abbreviated as TP0912 strain.

[0032] In this invention, SMP protein and AMD protein were selected as candidate vaccine antigens for immunoprotecting a mouse model infected with T. pyogenes. The recombinant proteins were obtained through a prokaryotic expression system. The recombinant proteins were mixed with aluminum hydroxide adjuvant in equal proportion and inoculated into mice. The protective effect was evaluated based on the specific antibody response, the expression of inflammatory cytokine genes, the expression of virulence factors, and the recovery of mice after bacterial challenge. The evaluated parameters included the expression level of virulence factors, the weight recovery of mice after bacterial challenge, the recovery of skin damage, the pathological observation results, and the survival rate. The research results showed that rSMP could provide effective immunoprotection, and rAMD had certain protectiveness, but it was still insufficient compared with rSMP.

[0033] Example 1 Experimental Methods

[0034] 1. Experimental Subjects:

[0035] Clean-grade female Kunming mice at 8 weeks old (weighing 18 - 20 g) were purchased from the Second Affiliated Hospital of Harbin Medical University, allowed to feed and drink freely, and acclimated to the laboratory conditions for one week before the experiment. The experimental protocol was approved by the Experimental Welfare and Ethics Committee of Northeast Agricultural University (NEAUEC - 202303142).

[0036] 2. Strains:

[0037] T. pyogenes (strain TP 0912) was isolated from the lungs of cattle (in Heilongjiang, China) suffering from pneumonia and cultured in Martin broth medium containing 5% fetal bovine serum (FBS) under aerobic conditions.

[0038] 3. Gene Cloning and Recombinant Plasmid Construction:

[0039] Using the GenBank database search, the sequences of the SMP gene (NCBI reference sequence: NZ_CP007519.1) and the AMD gene (NCBI reference sequence: WP_259145104.1) were found in the T. pyogenes genome. Genomic DNA was extracted from the T. pyogenes culture using the TIANamp Genomic DNA Kit (TIANGEN; Beijing; China) as a template. The PCR primers and restriction enzyme cleavage sites are shown in Table 1.

[0040] Table 1 Primer Sequences

[0041]

[0042]

[0043] In Table 1, the sequences corresponding to the restriction enzyme cleavage sites are underlined.

[0044] The PCR products were analyzed by agarose gel electrophoresis and purified using the TIANgel Purification Kit (Tiangen; Beijing; China). The purified DNA fragments were digested with restriction enzymes and then inserted into the pET-30a(+) plasmid vector. The recombinant plasmids were named pET-30a-SMP and pET-30a-AMD, respectively.

[0045] In subsequent studies, it was found that recombinant AMD (rAMD) had problems such as high molecular weight, complex structure, low expression level, and instability. To improve the expression level of rAMD and facilitate protein purification, in this study, the full-length gene of AMD was truncated into two genes, and the truncation primers are shown in Table 1. The plasmid construction method was the same as above. The recombinant plasmids were named pET-30a-AMD-1 and pET-30a-AMD-2, respectively, and were subjected to clone proliferation, plasmid extraction, and then DNA sequencing.

[0046] 4. Expression and purification of recombinant proteins:

[0047] The plasmids pET-30a-SMP, pET-30a-AMD-1, and pET-30a-AMD-2 were separately transformed into E. coli Rosetta(DE3) competent cells. They were spread on LB solid medium (containing 30 μg / mL kanamycin) and incubated at 37 °C in an inverted position for 9 - 12 h. Single colonies were picked and inoculated into liquid LB medium (containing 30 μg / mL kanamycin) and cultured overnight at 37 °C and 220 r / min. Then, the overnight culture was inoculated into a new LB liquid medium (containing 30 μg / mL kanamycin) at 2%. The culture was grown until the OD 600 reached 0.6 to 0.8, and then 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce expression. After 4 h of induction, the culture was centrifuged at 5000 r / min at 4 °C for 15 min, and the precipitate was collected for ultrasonic disruption and centrifugation to separate the precipitate and the supernatant. Samples were taken separately for SDS-PAGE electrophoresis to detect the expression of each recombinant protein.

[0048] The recombinant His-tagged proteins were purified according to the 6×His-tagged protein purification manual (Qiagen, USA) using the NI 2+ -NTA affinity chromatography method. At 4 °C, the protein solution was dialyzed against a gradient of PBS buffer containing 5% glycerol to remove urea from the purified protein solution. Finally, the dialyzed protein solution was concentrated using sucrose to obtain a high-concentration protein. The rSMP, rAMD-1, and rAMD-2 after dialysis and concentration were quantified using the BCA method and stored at -80 °C.

[0049] 5. Vaccination, bacterial challenge, and survival rate statistics:

[0050] Table 2 Immunization groups

[0051] Group Number of mice Immune component (100 μL) Group I 8 PBS Group II 8 Aluminum hydroxide + PBS Group III 8 Aluminum hydroxide + 50 μg rSMP Group IV 8 Aluminum hydroxide + 25 μg rAMD-1 + 25 μg rAMD-2 Group V 8 Aluminum hydroxide + 25 μg rSMP + 12.5 μg rAMD-1 + 12.5 μg rAMD-2

[0052] The immunization groups are shown in Table 2. Forty female Kunming mice were randomly divided into 5 groups, with 8 mice in each group. Among them, Group I and Group II served as control groups, and the immunization components were PBS and PBS + aluminum hydroxide adjuvant, respectively. Group III and Group IV were single-immunization groups, and the immunization components were rSMP + aluminum hydroxide adjuvant and rAMD (rAMD-1 + rAMD-2) + aluminum hydroxide adjuvant, respectively. Group V was a mixed-immunization group, and the immunization component contained rSMP + rAMD (rAMD-1 + rAMD-2) + aluminum hydroxide adjuvant. The antigen was mixed with the aluminum hydroxide adjuvant at a volume ratio of 1:1.

[0053] Each group of mice was subcutaneously inoculated with 100 μL of the vaccine on the back. The inoculation procedure was as Figure 1 shown. The mice were inoculated three times, with a two-week interval between each inoculation. On the 7th day, 21st day, and 35th day, blood samples were drawn from the tail vein, and the sera were collected and stored at -80 °C for detecting antibody titers.

[0054] On the 14th day after the third immunization, a bacterial challenge experiment was conducted on the mice. 2 × 10 9 CFU / mL of T. pyogenes (TP0912 strain) was injected subcutaneously into the abdomen. The mice were observed every 12 h for a 30-day observation period. The recovery and survival of the mice were recorded in detail, and a survival line graph was plotted.

[0055] 6. Histopathological observation:

[0056] On the 7th day after the bacterial challenge, the heart, liver, spleen, lung, and kidney tissues of the mice in each group were collected and fixed with 4% paraformaldehyde solution. The specimens were paraffin-embedded, sectioned, and stained with hematoxylin-eosin. The tissues were observed under a Nikon Eclipse Ti-U microscope at a magnification of 10 times. Then, images were acquired using NIS-Elements.

[0057] 7. Detection of mouse serum antibody levels:

[0058] To detect the specific antibody titer induced by antigens, an indirect enzyme-linked immunosorbent assay (ELISA) method was first established. The coating concentrations of each protein and the serum dilution ratios were preliminarily determined by checkerboard titration. The coating concentration of rSMP, rAMD-1 or rAMD-2 in the enzyme-linked immunosorbent assay (ELISA) plates (Nunc, USA) was 25 ng / well, and the coating volume was 100 μL / well. The coating solution used was carbonate buffer solution (pH = 9.6), and the plates were coated overnight at 4 °C. The next day, they were washed three times with PBST; then blocked with 5% skim milk containing 0.05% PBST at 37 °C for 2 h and washed three times with PBST; the serum was diluted with PBS, and the serum dilution ratios were 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, 1:2048000. After incubation at 37 °C for 1 h, they were washed 3 times with PBST; then incubated with 100 μL of HRP-labeled goat anti-mouse IgG antibody (ZSGB-BIO, China; diluted 1 / 5000 in 0.05% PBST) at 37 °C for 1 h and washed three times with PBST; then 50 μL / well of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added and developed for 20 min in the dark; 50 μL / well of 1 mol / L H2SO4 was added to terminate the reaction, and the OD of the antibody at 450nm the wavelength was detected using an enzyme-linked immunosorbent assay (ELISA) reader. The antibody titer of each group was expressed as the maximum dilution ratio of the immune group serum when the OD value of the immune group serum exceeded twice the OD value of the negative serum at the corresponding dilution ratio.

[0059] The serum before immunization of the mice was used as the negative serum, and the antibody titers of the serum after the first, second and third immunizations and 7 days after bacterial challenge were detected, and a line graph was plotted.

[0060] 8. Detection of mouse cytokines:

[0061] The Mouse IL-1β Valukine ELISA kit (Novus Biologicals, USA) and IL-10 Valukine ELISA kit (Novus Biologicals, USA) were used to detect the expression levels of two inflammation-related cytokines in the serum of mice.

[0062] Collect the heart, liver, spleen, lung, and kidney tissues of mice in each group on the 7th day after bacterial challenge. Use Trizol to extract total RNA from the organs according to the conventional method. Then, reverse transcribe the total RNA into cDNA using the ToloScript All-in-one RT EasyMix for qPCR kit (TOLOBIO, China). Real-time fluorescence quantitative PCR is performed using the 2×Q5 SYBR qPCR Master mix (Universal) kit (TOLOBIO, China) on the ABI 7500 Real-Time PCR system (Applied Biosystems, CA, USA). Select the gene encoding GAPDH as the internal reference. Calculate the relative expression level by the comparative Ct method. Use the 2 -ΔΔCT method to calculate the fold change of the immune group genes relative to the PBS group genes.

[0063] 9. Skin damage and body weight recovery after bacterial challenge:

[0064] For the surviving mice after bacterial challenge, quantitatively analyze the skin damage. Take pictures of the skin wounds with a camera at 4d, 8d, 12d, 16d, and 21d after bacterial challenge, and use the Image J image processing software to measure the wound size and calculate the percentage of wound healing area. The calculation formula is as follows:

[0065]

[0066] In the formula: A0 is the initial area of the wound, and A is the actual area of the wound at a certain time point.

[0067] Measure the body weight of the mice at a fixed time every day, monitor the dynamic changes in the body weight of mice in each group, take the body weight of the mice before bacterial attack as 100%, and draw a line graph of the body weight changes of mice in each group.

[0068] 10. Statistical analysis:

[0069] Use GraphPad Prism 9 (New York, USA) to analyze the data. At least three biological replicates were performed, and the analysis results are expressed as mean ± S.D. Since only 1 mouse survived in Group II and it did not have biological repeatability, the inter-group difference analysis for body weight and skin damage recovery did not consider Group II. One-way analysis of variance (ANOVA) was used to evaluate the inter-group differences. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0070] Results analysis of Example 2

[0071] 1. Preparation of immunogen:

[0072] Using the T. pyogenes genome as a template, a 1955-bp fragment was amplified by PCR. The recombinant plasmid pET-30a-SMP was constructed and identified by digestion with Xho I and Hind III, resulting in two bands of 5422 bp and 1955 bp, which were consistent with the expected results. The recombinant plasmid pET-30a(+)-AMD was constructed and identified by digestion with Kpn I and Hind III, resulting in two bands of 5422 bp and 3177 bp, which were consistent with the expected results. Sequencing confirmed that the target gene sequence was identical to the original sequence, thus completing the construction of the pET-30a-AMD recombinant plasmid. Using pET-30a(+)-AMD as a template, the nucleic acid fragments AMD-1 and AMD-2 amplified by PCR were 1548 bp and 1629 bp, respectively.

[0073] The positive recombinant plasmids pET-30a(+)-SMP, pET-30a(+)-AMD-1, and pET-30a(+)-AMD-2 were transformed into E. coli Rosetta(DE3) competent cells, induced for expression with IPTG (1 mmol / L), and purified by Ni-NTA affinity chromatography. SDS-PAGE analysis was performed on the purified rSMP, rAMD-1, and rAMD-2. Western Blot analysis was performed using a mouse His-tagged antibody, and the results are shown as follows. 2+ -NTA affinity chromatography to purify the proteins, and SDS-PAGE analysis was performed on the purified rSMP, rAMD-1, and rAMD-2. Western Blot analysis was performed using a mouse His-tagged antibody, and the results are as Figure 2 shown. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells. After induction with IPTG, the bacterial cells were collected and lysed. Recombinant His-tagged proteins were obtained by Ni-NTA affinity chromatography. Through Western Blot qualitative analysis and SDS-PAGE quantitative analysis, purified rSMP (90 kDa), rAMD-1, and rAMD-2 proteins (60 kDa) were obtained, respectively. 2+ -NTA affinity chromatography to obtain the recombinant His-tagged proteins. Through Western Blot qualitative analysis and SDS-PAGE quantitative analysis, purified rSMP (90 kDa), rAMD-1, and rAMD-2 proteins (60 kDa) were obtained, respectively.

[0074] 2. Determination of specific antibody levels after immunization and bacterial challenge:

[0075] Humoral immune responses play a crucial role in defending against bacterial infections. To evaluate the specific antibody responses induced by the vaccine, indirect ELISA assays were performed on the collected serum samples ( Figure 3 ).

[0076] After the third immunization, the level of specific antibodies in mice immunized with rSMP alone was lower than that in mice immunized with bivalent vaccine. On the contrary, the level of specific antibodies in mice immunized with rAMD alone was higher than that in mice immunized with bivalent vaccine. Among them, the level of specific antibodies in rAMD was higher than that in rSMP. On the 7th day after bacterial challenge, the levels of specific antibodies in both rSMP and rAMD increased significantly, and the levels in each group were consistent with the trend of the third immunization.

[0077] 3. Prognosis of mouse survival after bacterial infection:

[0078] After bacterial challenge, the skin at the injection site of bacteria in each group of mice showed varying degrees of damage. Image J was used to perform superposition analysis on the wounds at different times in each group ( Figure 4 as shown in A). The severity of skin damage in each group was in the order of GroupⅠ>GroupⅣ>GroupⅢ>GroupⅡ>GroupⅤ. The wound recovery rates of the surviving mice in each group at 4d, 8d, 13d, 16d, and 21d after bacterial challenge ( Figure 4 as shown in B). At 8d and 12d after bacterial challenge, compared with Group I, the wounds of Group III and Group V healed the fastest. At 16d after bacterial challenge, except for Group I, the wounds of mice in other groups had basically healed. At 21d after bacterial challenge, the mice in each group had basically recovered.

[0079] The weight recovery of the surviving mice after bacterial challenge was analyzed ( Figure 4 as shown in C). It was found that from 1 to 2 days after bacterial challenge, the weights of the mice in each group continued to decline. Among them, the weight of Group I decreased most severely, dropping to about 85%, and the decline in Group III and Group V was somewhat alleviated compared with other groups, only dropping to about 93%. From 2 to 4 days, the weights of each group tended to be stable. Among them, the weights of Group III and Group V showed the earliest upward trend. After 4 days, the weights of the mice in each group showed an upward trend. Among them, Group III and Group V first returned to normal weight.

[0080] 4. Histopathological observation:

[0081] Histopathological lesions were observed in mice 7 days after challenge with bacteria. In the mice of Group I and Group II, the main histopathological lesions were loose and irregularly arranged connective tissue in the heart valves, adhesion of red blood cells on the valve surface, necrosis of some cells in the valves, pyknosis of cell nuclei, and enhanced eosinophilia of cytoplasm; extensive dilation of venous blood vessels and erythrocyte filling in the liver tissue, showing congestion; a large number of vacuoles formed in the red pulp of the spleen, the structure of white pulp was damaged, and the clear marginal area around the lymph nodes became unclear. The pulmonary artery, vein, and alveolar wall capillaries were extensively dilated and filled with red blood cells; local tissue hemorrhage was observed, and a large amount of red blood cell aggregation was visible in the alveolar cavity; obvious hyperplasia of interstitial fibrous tissue in the kidney, unclear renal tubular structure, reduced number of renal tubular epithelial cells, and dilation of some blood vessels with erythrocyte filling, showing congestion. The use of monovalent or multivalent recombinant protein vaccines could alleviate the histopathological lesions caused by T. pyogenes infection to varying degrees. For example, the pathological changes in the lungs of Group III were alleviated. At the same time, the hepatocytes showed typical arrangement, and the renal cell nuclei were intact and stained well. The degree of pathological changes in the lungs of Group IV was similar to that of Group III, but liver lesions still existed( Figure 5 ).

[0082] 5. Detection of cytokine expression:

[0083] The mRNA levels of TNF-α, IL-1β, IL-6, IL-8, and IL-10 in the heart, liver, spleen, lungs, and kidneys of mice in each group on the 7th day after challenge with bacteria were analyzed. The expression levels of each gene were calculated by the 2 -ΔΔct method and normalized with GAPDH. Compared with other organs, the cytokine expression levels in the liver and lungs of each group were upregulated( Figure 6 A, B, C, D, E). Among them, all inflammatory factors in the liver and lungs of Group II were highly expressed, which might be the main reason for the high mortality rate in Group II. In the organs of Group III and Group V, the expression level of IL-8 was relatively high, and the expression levels of the remaining cytokines were lower than those of other groups.

[0084] Mouse IL-1β ELISA detection kit and mouse IL-10 ELISA detection kit were used to detect the expression levels of the two inflammatory factors in the sera of surviving mice on the 7th and 30th days after challenge with bacteria. In the ELISA detection results, IL-10 showed a high expression level in all five groups( Figure 6 G); on the 7th day after challenge with bacteria, IL-1β showed a high expression level in Group I, Group II, and Group IV, while the expression level was relatively low in Group III and Group V( Figure 6 F), indicating that mice in Group III and Group V received relatively good immune protection.

[0085] At the gene expression level, in the liver and lungs of the adjuvant group, except for IL-8, all other inflammatory factors had relatively high levels of expression, which was consistent with the relatively high mortality rate in the adjuvant group.

[0086] Some studies have found that T.pyogenes can induce macrophage pyroptosis and IL-1β release in mice through PLO, thereby causing inflammation. In this experiment, when comparing Group III with Group I, the expression level of IL-1β in Group III was relatively low ( Figure 6 B). By detecting the expression levels of IL-1β in the sera of each group, it was found that the expression levels in Group III were lower than those in other groups. Combining the detection results at these two levels, we inferred that SMP antibodies could more effectively resist the attack of T.pyogenes.

[0087] 6. Detection of anti-PLO antibody levels and mouse survival rate:

[0088] Virulence factors play a key role in the pathogenicity of bacteria. Among them, PLO, as the main virulence factor of T.pyogenes, while damaging the body, also acts as an immunogen to induce the body to produce an immune response and generate antibodies against PLO. In this study, rPLO was used as an antigen to perform an indirect ELISA detection on serum samples after bacterial attack. It was found that the PLO antibody levels in the SMP group were lower than those in the other four groups, while the PLO antibody titer in the AMD group was the highest. This indicates that SMP antibodies can effectively promote the clearance of T.pyogenes, reducing the possibility of the host immune system coming into contact with PLO, while the ability of AMD antibodies to promote the clearance of T.pyogenes is relatively poor ( Figure 7 in A).

[0089] The performance and survival rate of each group of mice after bacterial challenge were evaluated. It was found that within 12 h after bacterial challenge, symptoms such as ruffled fur, anorexia, and sluggish movement could be observed in all mice. In addition, the abdomen and back of the mice were sunken and they had difficulty walking. On the first day after bacterial challenge, except for the SMP group, mice in other groups died. The survival rates of the control group and the AMD group were both 57.14%. On the 30th day after bacterial invasion, the survival rate of the adjuvant control group was 14.28%, while the survival rates of the experimental groups containing rSMP immunogen were higher. The survival rates of the SMP group and the SMP + AMD group were 85.71% and 71.43% respectively ( Figure 7 in B).

[0090] In summary, in the present invention, a secreted protein (SMP) and a membrane protein (AMD) of T. pyogenes were selected to study their immunogenicity and protective efficacy. In animal experiments, the mice that received the two recombinant antigens did not show any discomfort, and there were no lesions around the injection site. Therefore, the recombinant antigens have good safety. The second and third immunizations induced stronger humoral and cellular immune responses, which had a significant protective effect against T. pyogenes challenge.

[0091] Analysis of inflammatory factors in the present invention revealed that in Group II, in the liver and lungs, except for IL-8, the expression levels of the other inflammatory factors were relatively high, which was consistent with the relatively low survival rate of Group II. Some studies have found that T. pyogenes can induce pyroptosis of mouse macrophages and the release of IL-1β through PLO, thus leading to inflammation. In this experiment, compared with Group I, the expression level of IL-1β in Group III was relatively low. By detecting the expression levels of IL-1β in the sera of each group, it was found that the expression levels in Group III were lower than those in other groups. Combining the detection results at these two levels, we inferred that the SMP antibody could resist T. pyogenes invasion.

[0092] The specific antibody response is a key indicator of the humoral immune response because this antibody plays a crucial role in protecting the host from bacterial infection. Analysis of antibody titers showed that the protein vaccine immunization could induce higher levels of antibody titers compared with the control group. Comparing the antibody titers against the two antigens, it was found that the specific antibody levels induced by the three immunizations with SMP were all lower than those with AMD. However, 7 days after the bacterial challenge, the specific antibody levels of SMP increased significantly. Previous exposure or vaccination might have generated good immune memory, enabling the rapid production of a large amount of antibodies when encountering the pathogen again. Therefore, although the baseline antibody levels were low, they could be rapidly enhanced when needed.

[0093] To further explore the protection of SMP and AMD to the host, the PLO antibody levels in the sera of mice in each group and the survival rate of mice were detected. It was found that in Group I, the PLO antibody level was relatively high, while in Group III, the PLO antibody level was relatively low. This indicated that the SMP antibody could help immunized mice effectively clear T. pyogenes in vivo. In contrast, in Group IV, the PLO antibody content was the highest among all groups, indicating that the AMD antibody had a relatively weak ability to clear T. pyogenes in immunized mice. The cumulative survival rate of mice in different immunized groups was higher than that of the control group. Moreover, the immunized groups containing the SMP component had a higher survival rate than the AMD group, indicating that SMP had more advantages in protecting the host and resisting T. pyogenes invasion.

[0094] The clinical symptoms of T. pyogenes are diverse, including subcutaneous abscesses, which often occur in livestock such as cattle and sheep. Through the bacterial challenge experiment on mice, typical skin damage symptoms caused by T. pyogenes appeared at the injection site. By observing the wound healing, the immunoprotective effect of the antigen was evaluated. The results showed that the wound healing rate of the immunized group was higher than that of the control group, and the wound healing rate of Group III was higher than that of Group IV. Therefore, both SMP antibody and AMD antibody can resist the invasion of T. pyogenes. Among them, the protective effect of SMP antibody is better than that of AMD antibody.

[0095] In summary, in this invention, recombinant SMP, AMD-1 and AMD-2 proteins were successfully obtained in the prokaryotic expression system. In the immunoprotective experiment of bacterial challenge, rSMP can provide effective immune protection, regulate the expression of inflammatory cytokine genes, and reduce histopathological lesions. Although rAMD also has certain protective effects, it is still insufficient compared with rSMP. rSMP can be used as a promising candidate antigen for anti-T. pyogenes vaccine. At the same time, as an enzyme protein, SMP is likely to play a certain biological function and have certain biological activities during the invasion of T. pyogenes into the body.

[0096] Therefore, the application of the above-mentioned SMP and AMD proteins in the preparation of anti-Arcanobacterium pyogenes vaccine in this invention can help the body produce a large amount of antibodies, resist the attack of T. pyogenes, reduce body damage, and improve the survival rate, which has important application value.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of SMP and AMD proteins in the preparation of a vaccine against Arcanobacterium pyogenes, characterized in that: It is applied to the preparation of a vaccine against Trueperella pyogenes. Use of SMP protein in the preparation of a vaccine against Trueperella pyogenes.

3. The application according to claim 2, characterized in that: The amino acid sequence of the SMP protein is shown as SEQ ID NO.

1. Use of AMD protein in the preparation of a vaccine against Trueperella pyogenes.

5. The application according to claim 4, characterized in that: The amino acid sequence of the AMD protein is shown as SEQ ID NO.

2.

6. A vaccine against Trueperella pyogenes, characterized in that: It contains the SMP protein with the amino acid sequence shown as SEQ ID NO.1 and / or the AMD protein with the amino acid sequence shown as SEQ ID NO.2 and an adjuvant.

7. The vaccine according to claim 6, wherein: The adjuvant is aluminum hydroxide.

8. A drug for preventing Trueperella pyogenes, characterized in that: It contains the SMP protein with the amino acid sequence shown as SEQ ID NO.1 and / or the AMD protein with the amino acid sequence shown as SEQ ID NO.2.

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