Application of SseB protein in preparation of cross immune homologous or heterologous serotype salmonella enteritidis vaccine

Abstract

The invention discloses an application of SseB protein in preparation of a vaccine for cross-immunization homologous or heterologous serotype salmonella enteritidis. Animal experiments are used for evaluating the immune protection capability of the salmonella enteritidis SseB protein, and results show that the salmonella enteritidis SseB protein has good immunoreactivity and can remarkably relieve clinical symptoms caused by homologous or heterologous serotype wild strains of salmonella enteritidis. The salmonella enteritidis SseB protein with the cross immune protection effect, provided by the invention, has the potential of developing a salmonella enteritidis subunit vaccine.

Description

Technical Field

[0001] The invention relates to the application of SseB protein in preparing cross-immune homologous or heterologous serotype enteritidis Salmonella vaccine, belonging to the application field of SseB protein. Background Art

[0002] Salmonella enteritidis (SE) is a Gram-negative bacillus. It is a non-spore, generally non-capsular, enteric bacillus with peritrichous flagella (capable of movement), and most of them also have pili. As an important foodborne and zoonotic pathogen, SE has become an important issue that needs to be urgently addressed in the field of global public health. It is closely related to a variety of gastrointestinal diseases. It can not only cause foodborne diseases in humans, but also cause high mortality in severe cases, posing a serious threat to human health. SE is widely distributed in the natural environment, has strong survival ability, and is easy to contaminate multiple links in the food chain such as poultry, livestock, eggs, and meat. Among these sources of pollution, contaminated poultry products are particularly prominent and become the main medium for the spread of diseases between farms and humans. According to statistics, about 30% of foodborne Salmonella cases worldwide can be attributed to the ingestion of contaminated poultry products, such as poultry meat and eggs, which have become the main channels for disease transmission due to their high consumption and susceptibility.

[0003] There are many types of vaccines on the market, including inactivated vaccines, subunit vaccines and live attenuated vaccines, each of which plays a different role in the prevention of animal and human diseases. Among them, although inactivated vaccines have been used in the prevention and control of Salmonella infection in poultry, their protective efficacy is often insufficient, mainly because they can only stimulate limited humoral immune responses, lack the synergistic effect of cellular immunity, and have poor immune persistence. In contrast, although live vaccines can stimulate strong immune responses and antibody production, their inherent virulence risks cannot be ignored and may cause unpredictable health problems. Subunit vaccines, with their single ingredients, high safety and good immunogenicity, have shown unique advantages in the poultry farming industry. Especially for Salmonella enteritidis, subunit vaccines have almost achieved perfect protection in preventing homologous serotype infections, but their protective power is significantly reduced when facing heterologous serotypes. Therefore, the development of a new Salmonella enteritidis vaccine that can simultaneously fight against multiple serotypes of Salmonella and has a broad protective efficacy has become an urgent need in the current field of poultry health management and food safety assurance.

[0004] SseB is a secreted protein belonging to the Espa superfamily, which is located on the surface of bacteria. As a key component of the type III secretion system (T3SS) transposon encoded by the Salmonella pathogenicity island 2 (Spi2), SseB shows a high degree of genetic conservation in Salmonella species. Previous literature reported that SseB protein, as a strong CD4 antigen, contains multiple immunodominant epitopes and can stimulate the host's immune response. In addition, recombinant SseB protein can increase the survival rate and number of days of mice after Salmonella typhimurium infection, and the bacterial load in the spleen is significantly reduced, which has a protective effect to a certain extent. All of this suggests the potential of SseB as a candidate antigen for Enteritidis Salmonella, but there are currently few studies exploring its cross-protection efficacy against homologous or heterologous serotypes of Enteritidis Salmonella. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of the type III secretion system protein SseB of Enteritidis Salmonella in the preparation of drugs or vaccines for cross-immunity to homologous or heterologous serotypes of Enteritidis Salmonella.

[0006] Technical solution: To solve the above technical problems, the present invention provides the use of SseB protein having an amino acid sequence as shown in SEQ ID NO.2 in the preparation of drugs or vaccines for cross-immunity to homologous or heterologous serotypes of Enteritidis Salmonella.

[0007] Wherein, the homologous serotype Enteritidis Salmonella includes Enteritidis Salmonella CMCC(B)50041 or Enteritidis Salmonella CVCC3949.

[0008] Wherein, the heterologous serotype Enteritidis Salmonella includes Salmonella typhimurium UK-1.

[0009] The present invention also provides the use of the SseB protein with an amino acid sequence as shown in SEQ ID NO.2 in the preparation of a drug or vaccine for preventing homologous serotype Enteritidis Salmonella.

[0010] Wherein, the homologous serotype Enteritidis Salmonella includes Enteritidis Salmonella CMCC(B)50041 or Enteritidis Salmonella CVCC3949.

[0011] The present invention also provides a vaccine, which contains the SseB protein with an amino acid sequence as shown in SEQ ID NO.2.

[0012] Wherein, the vaccine also contains a vaccine adjuvant.

[0013] Among them, the vaccine increases the expression levels of IFN-γ, IL-4, and IL-17a mRNA.

[0014] The present invention discloses a SseB protein of the type III secretion system of Enteritidis Salmonella with cross-immune protection. The sseB gene fragment is obtained by PCR, and the recombinant SseB protein is prepared by a prokaryotic expression method; the immune protection is evaluated by animal experiments. The results show that SseB protein can significantly reduce the clinical symptoms caused by Enteritidis Salmonella CMCC (B) 50041, homologous serotype CVCC3949 Enteritidis Salmonella and Salmonella typhimurium UK-1 after being challenged with the virus. The SseB protein screened by the present invention, which has an immune protection effect on Salmonella Enteritidis in chickens, has the potential to be used for the development of a subunit vaccine for Salmonella Enteritidis in chickens.

[0015] (1) Amplify the sseB gene of Salmonella enteritidis and construct the expression plasmid pET28a-sseB.

[0016] (2) The expression plasmid pET28a-sseB was transformed into the expression engineering bacteria BL21 (Rosseta) to obtain the expression strain BL21 (pET28a-sseB).

[0017] (3) SseB protein was expressed in Escherichia coli and purified by High-Affinity Ni-NTAResin FF.

[0018] (4) Use Western blot to identify whether SseB protein expression is correct.

[0019] (5) Detect the specific immune response induced by immunization with the SseB protein subunit vaccine m of Salmonella enteritidis.

[0020] The present invention also detects the cross-immunity protection efficacy of the SseB protein subunit vaccine of Salmonella enteritidis on SPF chickens.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention relates to a SseB protein of Enteritidis Salmonella with cross-immune protection. The sseB gene fragment is obtained by PCR amplification, and the recombinant SseB protein is prepared by a prokaryotic expression method; and its immune protection is evaluated by animal experiments. The results show that the SseB protein of Enteritidis Salmonella has good immunoreactivity and can significantly reduce the clinical symptoms caused by wild strains of homologous or heterologous serotypes of Enteritidis Salmonella. The SseB protein of Enteritidis with cross-immune protection disclosed by the present invention has the potential to be used for the development of subunit vaccines for Enteritidis Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 PCR identification of BL21 (pET28a-sseB) bacterial solution (a) and verification of pET28a-sseB plasmid by double restriction enzyme digestion (b);

[0023] Figure 2 SDS-PAGE identification of SseB protein expression; Lane 1: supernatant sample after induction and disruption; Lane 2: precipitate sample after induction and disruption; Lane 3: whole bacteria after induction; Lane 4: whole bacteria without induction;

[0024] Figure 3 This is the SDS-PAGE identification of the purified SseB protein, wherein lane 1: column equilibrium solution; lane 2: purified flow-through sample; lanes 3-9: purified eluate samples;

[0025] Figure 4 Western blot identification of SseB protein, including 1: BL21 (pET28a empty plasmid), 2: BL21 (pET28a-sseB);

[0026] Figure 5 The immune response induced by SseB antigen: a: ELISA was used to measure the serum IgY titer; b: CCK-8 method was used to detect the proliferation response of chicken peripheral blood lymphocytes; ce: IFN-γIL-4, IL-17A mRNA expression in lymphocytes was measured (***p<0.001);

[0027] Figure 6 The cross-protection of the candidate antigen SseB protein against homologous and heterologous serotypes of Enteritidis Salmonella: a, e are the survival rates of chickens in each group after challenge with CMCC(B)50041 and CVCC3949 strains to determine the protective effect; bd are the colonization of CMCC(B)50041 and SseB in chicken organs and tissues; log CFU / g or log CFU were used to evaluate bacterial colonization in the liver, spleen and cecum (***p<0.01);

[0028] Figure 7 is the cross-protection of the candidate antigen SseB protein against homologous and heterologous serotypes of Enteritidis Salmonella; d is the survival rate of each group of chickens after challenge with UK-1 strain to determine the protective effect; ac, eg are the colonization of CVCC3949, SseB, and UK-1 in chicken organs and tissues; log CFU / g or log CFU was used to evaluate the bacterial colonization of the liver, spleen, and cecum (***p<0.01). DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0030] Example 1: Cloning and prokaryotic expression of the sseB gene of Salmonella enteritidis

[0031] The gene sequence of sseB was extracted from the complete gene sequence of Salmonella enteritidis (CMCC(B)50041), and the sequence is shown in SE QID NO.1: ATGTCTTCAGGAAACATCTTATGGGGAAGTCAAAACCCTATTGTGTTT. The sseB gene without a signal peptide sequence was amplified using a BamH I site and a Hind III site, and the gene was PCR amplified using the genomic DNA of Salmonella enteritidis as a template. The PCR product was subcloned into the pET-28a vector with a His tag. The recombinant plasmid was named pET28a-sseB. The recombinant plasmid was then transformed into the engineered bacterium Escherichia coli BL21, and the clones were picked and identified by PCR and sequencing. The PCR reaction system was 25 μL, and the configuration was as follows: 12.5 μL of 2×Taq Master Mix (Dye Plus) enzyme, 8.5 μL of ddH2O, 1 μL of upstream and downstream primers, and 2 μL of template. The common Taq enzyme PCR reaction conditions used for identification are as follows: 94°C for 5 min; 94°C for 40 sec, 55°C for 30 sec, 72°C for 1 min, 35 cycles; 72°C for 10 min. The correct clone was identified as the positive clone and named BL21 (pET28a-sseB).And it was confirmed by double enzyme digestion and DNA sequencing, where the enzyme digestion system was 20μL, BamH I 1μL, HindⅢ1μL, 10×K buffer 2μL sseB plasmid 5μL (concentration was 200μg / mL), and dd H2O was used to make up to 20μL. PCR results are as follows. Figure 1 As shown in A, the double enzyme digestion identification results are as follows Figure 1 B. Among them, the PCR primers were pET28a universal T7 primers (F: 5'-TAATACGACTCACTATAGGG-3', R: 5'-GCTAGTTATTGCTCAGC GG-3') and a fragment of the expected size was amplified, about 750 bp, confirming that the BL21 (pET28a-sseB) strain was successfully obtained. Figure 1 In A, 1: BL21 (pET28a-sseB), +: BL21 (pET28a empty plasmid), -: dd H20; Figure 1 In B, 1: pET28a-sseB, 2: pET28a, 3: dd H20.

[0032] The steps for identifying whether the SseB protein is expressed are as follows:

[0033] (1) The obtained recombinant Escherichia coli BL21 (pET28a-sseB) was cultured in 100 mL LB liquid medium until OD 600 When the pH was 0.5, isopropylthiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the expression was induced on a shaker at 37°C for 4 hours.

[0034] (2) The BL21 (pET28a-sseB) cells were collected by centrifugation at 8000 rpm / min for 10 min and resuspended in 16 mL PBS (formula: KCl 0.2 g, NaCl 8 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, 1000 mL distilled water, pH 7.6).

[0035] (3) After disrupting the cells using an ultrasonic disruptor, centrifuge at 8000 rpm / min for 10 min at 4°C to collect the supernatant, and resuspend the precipitate in 16 mL of PBS for later use.

[0036] (4) Take 80 μL of the supernatant and precipitate resuspension obtained in step (3), add 20 μL of loading buffer (1 mL of 1M Tris-HCl (pH 6.8), 0.31 g of 200 mM DDT, 0.4 g of 4% SDS, 0.02 g of 0.2% bromophenol blue, 2 mL of 20% glycerol, 7 mL of ultrapure water), mix well, and boil in 100°C boiling water for 10 min. Use 12% SDS PAGE gel electrophoresis to identify whether the protein is expressed. The results are as follows. Figure 2 As shown, the target band appeared at about 21 kDa in both the supernatant and the precipitate.

[0037] Example 2: Purification of Salmonella Enteritidis sseB protein

[0038] The supernatant sample obtained after the crushing in Example 1 was further purified using High-Affinity Ni-NTAResin, and the specific method is as follows:

[0039] (1) Add 5 mL of Ni-NTA metal chelated His protein purification medium (purchased from GenScript Corporation) to the affinity chromatography column;

[0040] (2) Add 15 mL of ddH2O to the affinity chromatography column for washing;

[0041] (3) The supernatant sample obtained after the crushing in Example 1 was filtered through a 0.45 μm pore size filter and added to an affinity chromatography column to collect the flow-through;

[0042] (4) Add equilibration buffer (60 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) to equilibrate the column;

[0043] (5) Add 30 mL of washing solution (60 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0) to wash away impurities and collect the washing solution;

[0044] (6) Add 8 mL of elution buffer (60 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0) to elute the target protein and collect the eluate in 7 tubes;

[0045] (7) Take 80 μL of each of the flow-through, wash solution, and eluate and add 20 μL of loading buffer and boil for 10 min;

[0046] (8) Prepare 12% SDS-PAGE polyacrylamide gel, add the sample treated in step (7) to the well for electrophoresis. After the electrophoresis is completed, remove the gel and stain it with Coomassie Brilliant Blue overnight, then decolorize it to identify whether SseB is already present in the eluate. The results showed that there was a high-purity SseB protein in the eluate, with a size of 21 kDa ( Figure 3 );

[0047] (9) The eluate containing the target protein was dialyzed and concentrated through a 10 kDa ultrafiltration tube, and the buffer was replaced with PBS to obtain a SseB protein suspension dissolved in PBS.

[0048] The amino acid sequence of the purified recombinant SseB protein is shown in SEQ ID NO.2: MSSGNILWGSQNPIVFKNSFGVSNADTGSQDDLSQQNPFAEGYGVLLILLMVIQAIANNKFIEVQKNAERARNTQEKSNEMDEVIAKAAKGDAKTKEEVPEDVIKYMRDNGILIDGMTIDDYMAKYGDHGKLDKGGLQAIKAALDNDANRNTDLMSQGQITIQKMSQELNAVLTQLTGLISKWGEISSMIAQKTYS

[0049] Example 3: Identification of the sseB protein of Salmonella enteritidis

[0050] The purified protein obtained in Example 2 was further identified by Western blot. The purified recombinant protein was dissolved in sample buffer and subjected to electrophoresis. The protein was electrotransferred to a nitrocellulose membrane using a microdialyzer (Bio-Rad). The membrane was incubated overnight in a blocking buffer at 4°C, washed with PBST, and His-Tag (Boster M30975) was used as the primary antibody and HRP-labeled goat anti-mouse IgG (Boster BA1050) was used as the secondary antibody. The primary antibody dilution was 1:3000 and the secondary antibody dilution was 1:5000. The enhanced chemiluminescence (ECL) substrate was used to detect the signal to further verify that the sseB protein of Salmonella Enteritidis (CMCC (B) 50041) was correctly expressed ( Figure 4 ).

[0051] Example 4: Determination of the cross-immunity protection efficacy of the SseB protein subunit vaccine of Salmonella enteritidis

[0052] In order to evaluate the immune protection of the candidate antigen SseB, experimental one-day-old SPF chickens were randomly divided into 6 groups, namely, blank control group, CMCC(B)50041 challenge group, CVCC3949 challenge group, UK-1 challenge group and SseB protein immunization group. SseB protein immunization group: the expressed recombinant protein was diluted with PBS at a volume ratio of 1:4 (the protein concentration was measured to be 4μg / μL, diluted to 1μg / μL), mixed with Montanide TM ISA206VG adjuvant (manufacturer: SEPPIC) at a volume ratio of 1:1, and then fully emulsified to prepare a subunit vaccine. The vaccine preparation containing 100μg SseB protein was used for intramuscular injection (im) of the leg of SPF chickens for immunization, with the first vaccination on day 0 (1 day old), and then booster immunization on day 14. Blank control group: 200μL of a mixture of PBS and Montanide TM ISA 206VG adjuvant (mixed at a volume ratio of 1:1) was injected. To determine the antibody response, blood samples were collected from the vaccinated group and the blank control group via the wing vein every week until 2 weeks after the final immunization. Serum was separated and tested for antibodies by iELISA as well as cytokine detection. qPCR system The specific response induced by SseB protein after immunization is shown in Figure 2. Figure 5 As shown in a, the antibody reached a high level 3 weeks after booster immunization; and compared with the control group (cells without protein stimulation), the SseB antigen protein stimulated peripheral blood lymphocytes (PBMCs) in the immunized group and significantly increased the cell proliferation level ( Figure 5 b); Cytokines were detected by qPCR, where the qPCR reaction system: 10μL 2×Taq Pro Universal SYBR qPCR Master Mix, 0.4μL Primer 1 (10μM), 0.4μL Primer 2 (10μM), 2μL Template DNA / cDNA, 7.2μL ddH2O. Amplification program: 95℃ pre-denaturation for 30s, 95℃ denaturation for 10s, 60℃ annealing for 30s (40 cycles). The results show ( Figure 5 ce) SseB protein immunization can cause an increase in IFN-γ, IL-4, and IL-17a mRNA levels to a certain extent. The results show that SseB protein has good immunogenicity and can activate humoral and cellular immune responses in chickens. Among them, Figure 5 The 1640 group was 1640 culture medium, i.e., a negative control without any protein stimulation, and conA was concanavalin A as a positive control.

[0053] Two weeks after booster immunization, six 28-day-old chickens were randomly selected from the subunit vaccine group (SseB protein immunization group) and the challenge group and injected intramuscularly with 10LD50 The chickens were challenged with logarithmic phase Salmonella enterica strains CMCC(B)50041, Salmonella enterica CVCC3949 and Salmonella typhimurium UK-1 (CMCC(B)50041 was purchased from the China Food and Drug Inspection Institute, and CVCC3949 and UK-1 were purchased from the China Veterinary Drug Administration) (CFU / bird). Chickens that were not challenged with the virus served as the control group and were injected with the same dose of PBS. The test was continued for 14 days, and the clinical symptoms and deaths of the chickens were recorded every day, and a survival curve was constructed. The remaining chickens were euthanized 7 days after the challenge (dpc) to collect the liver, spleen and intestines for the determination of bacterial load. The results are as follows Figure 6 and Figure 7 As shown, the SseB protein has the highest protection efficiency against the homologous serotype CMCC(B)50041 strain (83%), the protection efficiency against the homologous serotype CVCC3949 strain is 66.7%, and the protection efficiency against the heterologous serotype strain Salmonella typhimurium UK-1 is 33.3% ( Figure 6 a, 6e, 7e). Bacterial organ burden results are as follows Figure 6 and Figure 7 As shown in (6b-d, 7a-c, 7e-g), SseB protein immunization can reduce the number of bacteria in the liver, spleen and cecum to varying degrees, and has a good protective effect. In conclusion, SseB as a subunit vaccine has good immunogenicity, can stimulate the body to produce high levels of humoral and cellular immune responses, and has different degrees of protection against homologous and heterologous serotypes of Salmonella infection.

Claims

1. Use of the SseB protein having an amino acid sequence as shown in SEQ ID NO.2 in the preparation of a drug or vaccine for cross-immunization with homologous or heterologous serotypes of Enteritidis Salmonella.

2. The application according to claim 1, characterized in that: The homologous serotype Enteritidis Salmonella includes Enteritidis Salmonella CMCC(B)50041 or Enteritidis Salmonella CVCC3949.

3. The application according to claim 1, characterized in that: The heterologous serotypes of Enteritidis include Salmonella typhimurium UK-1.

4. Use of the SseB protein having an amino acid sequence as shown in SEQ ID NO.2 in the preparation of a drug or vaccine for preventing homologous serotype Enteritidis Salmonella.

5. The use according to claim 4, characterized in that: The homologous serotype Enteritidis Salmonella includes Enteritidis Salmonella CMCC(B)50041 or Enteritidis Salmonella CVCC3949.

6. A vaccine, characterized in that It contains the SseB protein with the amino acid sequence shown in SEQ ID NO.

2.

7. The vaccine according to claim 6, characterized in that The vaccine also contains a vaccine adjuvant.

8. The vaccine according to claim 6, characterized in that The vaccine increases the expression levels of IFN-γ, IL-4, and IL-17a mRNA.

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