Salmonella polyepitope protein, encoding gene and use thereof in preparing salmonella subunit vaccine

Abstract

The application discloses a Salmonella multi-epitope protein, a coding gene and application of the Salmonella multi-epitope protein in preparation of a Salmonella subunit vaccine, and relates to the field of biotechnology.The amino acid sequence of the Salmonella multi-epitope protein is shown as SEQ ID NO.2.The nucleotide sequence of the coding gene of the Salmonella multi-epitope protein is shown as SEQ ID NO.1.The Salmonella subunit vaccine prepared by using the Salmonella multi-epitope protein has the advantages of safety and stability, low cost, no toxicity to the body, and the ability to simultaneously cause the body to produce humoral immunity and cellular immunity, and the like, and can not only produce a preventive protection effect on Salmonella enteritidis infection, but also can play a cross protection effect on Salmonella typhimurium infection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to Salmonella multiepitope proteins, their encoding genes, and their application in the preparation of Salmonella subunit vaccines. Background Technology

[0002] Salmonella enteritidis is a facultative intracellular pathogen that can be carried asymptomatically in the intestines or gallbladder of livestock and poultry, causing gastroenteritis and becoming one of the leading causes of foodborne illnesses in humans worldwide. Antibiotic treatment is the primary means of combating Salmonella enteritidis infection; however, given the increasing prevalence of multidrug-resistant Salmonella in animals and the dangers of drug residues, the development of safe and effective Salmonella vaccines is receiving increasing attention.

[0003] Currently, Salmonella vaccines under research mainly include inactivated vaccines, live attenuated vaccines, and subunit vaccines. Inactivated vaccines do not induce cellular immunity and have poor immunogenicity. Live attenuated vaccines are the most favored because they can stimulate cellular and humoral immunity in the host and provide a long-term protective immune response, but they have the potential for virulence reversion and it is difficult to distinguish between infected and immunized animals. Compared with whole-cell vaccines, subunit vaccines are safer, but they often need to be used with adjuvants to improve immunogenicity. On the other hand, the Salmonella genome is highly variable, so conventional subunit vaccines based on virulence factors may have limited applicability and cannot provide protection against heterologous strains, requiring new vaccine development strategies.

[0004] Multiepitope vaccines utilize minimal immunogenic fragments to trigger effective immune responses against specific pathogens. Compared to the entire homologous protein, epitopes can induce immune responses more directly. While inducing humoral and cellular immunity, they overcome the limitations of traditional live attenuated vaccines, such as reversion, and offer advantages such as good safety, low cost, ease of use, and easy differentiation between infected and immunized animals. Furthermore, proteins expressed by the pathogen at different stages and in different environments can be used as candidate antigens and combined in multiepitope vaccines to improve their universal applicability. Based on this, this invention aims to develop a novel Salmonella enteritidis multiepitope subunit vaccine to provide effective immunoprotection against Salmonella infection. Summary of the Invention

[0005] The purpose of this invention is to provide a Salmonella multiepitope protein, its encoding gene, and its application in the preparation of Salmonella subunit vaccines, thereby addressing the problems existing in the prior art. The Salmonella subunit vaccine prepared using this Salmonella multiepitope protein has advantages such as safety and stability, low cost, non-toxicity to the body, and the ability to simultaneously induce humoral and cellular immunity. It not only provides preventive protection against Salmonella enteritidis infection but also offers cross-protection against Salmonella typhimurium infection.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a Salmonella multiepitope protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides a gene encoding the above-mentioned Salmonella multiepitope protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] The present invention also provides a recombinant expression vector comprising the above-described coding gene.

[0010] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0011] Furthermore, the recombinant host cell is *Escherichia coli* FLPD;

[0012] The *Escherichia coli* FLPD strain was deposited on January 17, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2024132.

[0013] The present invention also provides the application of the above-mentioned encoding gene, recombinant expression vector or recombinant host cell in the preparation of Salmonella multiepitope protein.

[0014] The present invention also provides the application of the above-mentioned Salmonella multi-epitope protein in the preparation of Salmonella subunit vaccines.

[0015] Furthermore, the Salmonella is Salmonella enteritidis and / or Salmonella typhimurium.

[0016] The present invention also provides a Salmonella subunit vaccine, the active ingredient of which includes the above-mentioned Salmonella multiepitope protein.

[0017] Furthermore, the Salmonella subunit vaccine also includes a vaccine adjuvant.

[0018] The present invention discloses the following technical effects:

[0019] This invention predicts and screens B-cell epitopes and antigenic epitopes that can bind to MHC class I and MHC class II molecules from FliC and SipD proteins. A Salmonella multi-epitope protein FLPD is prepared through linker fusion expression. A Salmonella subunit vaccine is then prepared using this FLPD protein. This Salmonella subunit vaccine has advantages such as safety, stability, low cost, non-toxicity to the body, and the ability to simultaneously induce humoral and cellular immunity. It not only provides preventive protection against Salmonella enteritidis infection but also offers cross-protection against Salmonella typhimurium infection. Furthermore, this method does not require microbial culture and can also provide effective protection against some variant strains. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the technical route of the present invention;

[0022] Figure 2 The plasmid map of the target plasmid pET-28b(+)-FLPD;

[0023] Figure 3 The results show the tertiary structure and molecular docking of the FLPD protein; where A is the tertiary structure of the FLPD protein; B is the docking model of the FLPD protein with MHC-I molecules; C is the docking model of the FLPD protein with MHC-II molecules; and D is the docking model of the FLPD protein with the TLR-4 receptor molecule.

[0024] Figure 4 This is a comparison diagram of sequencing results;

[0025] Figure 5 The image shows the SDS-PAGE results of FLPD protein expression after induction. Lane A represents the protein expression results induced by different IPTG concentrations; lane M represents the protein marker; lane 1 shows the results of pET-28b(+)-FLPD without induction; lanes 2 and 3 show the induction results of empty vector pET-28b(+) at an IPTG concentration of 1.0 mmol / L; lanes 4-9 show the results of pET-28b(+)-FLPD induced by different IPTG concentrations of 0.3 mmol / L, 0.5 mmol / L, 0.8 mmol / L, 1.0 mmol / L, and 1.5 mmol / L. mol / L and 2.0 mmol / L; B shows the protein expression results induced at different times: lane M is the protein marker; lanes 1 and 2 show the induction results of empty vector pET-28b(+) at IPTG concentration of 1.0 mmol / L, 37℃ for 4 h; lanes 3-6 show the results of pET-28b(+)-FLPD after induction for 7, 6, 5, and 4 h, respectively; C shows the protein soluble expression results: lane M is the protein marker; lane 1 shows the whole bacteria induced by pET-28b(+)-FLPD at 37℃, 4 h, and 1.0 mmol / L; lane 2 shows the supernatant after sonication, and lane 3 shows the precipitate after sonication;

[0026] Figure 6Figure 1 shows the SDS-PAGE and Western blot results of FLPD protein purification. In this figure, A represents the SDS-PAGE results of the purified protein: lane M is the protein marker; lanes 1-6 represent whole cells before autoclaving, whole cells after autoclaving, supernatant, flow-through buffer, flow-through buffer, and precipitate, respectively; lanes 7-8 are the washing buffer; lanes 9-10 are 50 mmol / L imidazole elution buffer; lanes 11-13 are 100 mmol / L imidazole elution buffer; lane 14 is 2... Lanes 15-18 contain 200 mmol / L imidazole elution buffer; lanes 19-22 contain 250 mmol / L imidazole elution buffer; lane 23 contains 300 mmol / L imidazole elution buffer; lane 24 contains 500 mmol / L imidazole elution buffer; lanes 25-28 contain whole cells, supernatant, supernatant, and precipitate after autoclaving; lane B is for protein Western blot validation; lane M is for protein marker; lane 1 contains purified protein FLPD.

[0027] Figure 7 The image shows the protein purity test results. Lane M represents the protein marker; lanes 1-7 contain BSA standard proteins at concentrations of 0.5 mg / mL, 0.4 mg / mL, 0.3 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, and 0.025 mg / mL, respectively; lane 8 contains 250 mmol / L imidazole elution buffer; lane 9 contains fusion tubes with 200 mmol / L and 250 mmol / L imidazole elution buffers; lane 10 contains 300 mmol / L imidazole elution buffer; lane 11 contains 200 mmol / L imidazole elution buffer; lane 12 contains 200 mmol / L imidazole elution buffer; lane 13 contains 250 mmol / L imidazole elution buffer; and lane 14 contains 250 mmol / L imidazole elution buffer.

[0028] Figure 8 Standard curves for protein concentration versus A562 (A) and protein concentration versus grayscale value (B) are shown.

[0029] Figure 9 A statistical graph showing the changes in body weight of mice immunized with different doses;

[0030] Figure 10 A statistical chart showing the titer of specific IgG antibodies in the serum of immunized mice;

[0031] Figure 11 A statistical graph showing the expression levels of mouse serum cytokines IFN-γ (A), IL-4 (B), IL-6 (C), and IL-12 (D);

[0032] Figure 12The figures show the survival curves of mice after challenge with the virus; where A is the survival curve of Salmonella enteritidis after the second immunization; B is the survival curve of Salmonella typhimurium after the second immunization; C is the survival curve of Salmonella enteritidis after the third immunization; and D is the survival curve of Salmonella typhimurium after the third immunization.

[0033] Figure 13 This is a statistical chart of bacterial load in mice;

[0034] Figure 14 A statistical chart showing the changes in the weight of chicks;

[0035] Figure 15 A statistical chart showing the titer of specific IgG antibodies in the serum of immunized chicks;

[0036] Figure 16 This is a survival curve for chicks. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] The technical route of the present invention is as follows: Figure 1 As shown, the research involves the following content:

[0043] 1) Based on previous research on effective antigenic proteins of Salmonella, suitable candidate antigenic proteins in Salmonella enteritidis were screened, namely Salmonella enteritidis flagella protein FliC and type III secretion system protein SipD.

[0044] 2) Download the FASTA sequences of Salmonella enteritidis FliC (KKE15205.1) and SipD (KKE05642.1) proteins from NCBI.

[0045] 3) Predict the antigenicity, sensitization, hydrophilicity, signal peptide region, and transmembrane structure of candidate antigens FliC and SipD to ensure that the selected protein is non-toxic, non-sensitizing, hydrophilic, and free of signal peptide region and transmembrane structure.

[0046] 4) Predict and screen B cell epitopes and antigenic epitopes that can bind to MHC class I and MHC class II molecules from FliC and SipD proteins.

[0047] 5) The selected antigen epitope sequences were expressed by adapter fusion to prepare recombinant protein FLPD.

[0048] 6) Mix recombinant protein FLPD with adjuvant at a certain concentration and in an equal proportion to obtain a Salmonella multi-epitope subunit vaccine.

[0049] 7) The prepared FLPD Salmonella multi-epitope vaccine was used to conduct immunization and challenge protection tests on SPF BALB / c mice. The protective effect of the FLPD vaccine was evaluated through a series of tests, including vaccine safety, specific antibody titer, cytokine level, survival curve, organ bacterial load, and histopathological evaluation.

[0050] 8) Immunization and challenge protection tests were conducted on SPF White Leghorn chicks using the prepared FLPD Salmonella multiepitope vaccine. Commercially available live attenuated Salmonella enteritidis vaccine (Sm24 / Rif12 / Ssq strain) was used as a positive control for immunizing chicks. A series of tests, including changes in chick weight, specific antibody levels, and protection rates, were conducted to evaluate the immunoprotective efficacy of the FLPD multiepitope vaccine against Salmonella enteritidis-infected chicks and to preliminarily explore the cross-protective effect of FLPD against other Salmonella serotypes.

[0051] The experimental materials used in the following examples are as follows:

[0052] Two hundred and twenty SPF-grade, six-week-old BALB / c mice were provided by the Experimental Animal Center of Huazhong Agricultural University. The first batch of sixty mice was used to determine the median lethal dose (LD50) of Salmonella enteritidis and Salmonella typhimurium in mice. 50 Experiment. A second batch of 160 mice was used for vaccine immunization research. They were housed at the Experimental Animal Center of Huazhong Agricultural University. Ethics Number: HZAUMO-2024-0030.

[0053] Two batches of SPF White Leghorn chicks: the first batch consisted of 3-week-old chicks, and the second batch consisted of 1-day-old chicks, both purchased from Boehringer Ingelheim Viton Biotechnology Co., Ltd. in Beijing. The first batch of SPF chicks consisted of 20 chicks, intended for use against Salmonella enteritidis and LD50 in chicks. 50 Experiment. A second batch of 35 SPF chicks were used for vaccine immunization research. They were raised in stainless steel poultry isolators at the Department of Veterinary Medicine, Huazhong Agricultural University, with free access to water and SPF chick feed. The rearing temperature was maintained at 36℃~37℃ for the first week, then decreased by 1℃ daily until reaching 30℃, while the room temperature was maintained at 24℃. Ethics Certificate No.: HZAUCH-2024-0005.

[0054] The experimental research of this invention was conducted in accordance with the guidelines stipulated in the "Regulations on the Management of Laboratory Animals in China" (1988) and the "Regulations on the Management of Laboratory Animals in Hubei Province" (2005). In this invention, all work was conducted to treat laboratory animals ethically and to minimize their suffering.

[0055] Luria-Bertani (LB) liquid medium: Accurately weigh 25.00 g of LB liquid medium, add 1000 mL of distilled water, autoclave at 121 °C for 15 min, and set aside.

[0056] 2% agarose gel: Weigh 1.2g of agarose, heat and dissolve it in 60mL of 1×TAE buffer. After cooling to about 50℃, add nucleic acid dye, mix thoroughly and pour into a gel plate. Use after it has solidified completely.

[0057] Kanamycin (50 mg / mL): Place 1 g of powder sample in a 50 mL tube, add 20 mL of sterile water in a clean bench, filter the solution into a 2 mL centrifuge tube using a syringe, and store in a refrigerator at -20°C.

[0058] IPTG (isopropylthio-β-D-galactopyranoside): Dissolve 2g of IPTG in 8mL of distilled water, bring the volume to 10mL with distilled water, filter through a 0.22μm filter for sterilization, dispense into 1mL portions, and store at -20℃. The prepared IPTG concentration is 0.8mmol / mL.

[0059] Lysis Buffer: Accurately weigh 7.80 g of dihydrate and sodium dihydrogen phosphate, 17.54 g of sodium chloride, and 0.68 g of imidazole, dissolve them in 900 mL of double-distilled water, add double-distilled water to bring the volume to 1 L, adjust the pH to 8.0, filter using a 0.22 μm aqueous phase filter membrane, and store at 4 °C.

[0060] Wash Buffer: Accurately weigh 7.80 g of dihydrate and sodium dihydrogen phosphate, 17.54 g of sodium chloride, and 6.80 g of imidazole, dissolve them in 900 mL of double-distilled water, add double-distilled water to bring the volume to 1 L, adjust the pH to 8.0, filter using a 0.22 μm aqueous phase filter membrane, and store at 4 °C.

[0061] Elution Buffer: Accurately weigh 7.80 g of dihydrate and sodium dihydrogen phosphate, 17.54 g of sodium chloride, and 17.0 g of imidazole, dissolve them in 900 mL of double-distilled water, add double-distilled water to bring the volume to 1 L, adjust the pH to 8.0, filter using a 0.22 μm aqueous phase filter membrane, and store at 4 °C.

[0062] Coomassie Brilliant Blue Staining Solution: Accurately weigh 1g of Coomassie Brilliant Blue R-250 and place it in a 1L beaker. Measure and add 250mL of isopropanol, 100mL of glacial acetic acid, and 650mL of deionized water. Stir well and store at room temperature.

[0063] Coomassie Brilliant Blue Decolorizing Solution: Measure 40 mL of acetic acid (glacial acetic acid), 180 mL of methanol, and 180 mL of deionized water, mix thoroughly, and then use.

[0064] SEQ ID NO.1:

[0065] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCAGGATGAAATTCAGCAGCGTCTGGAAGAAATTGATCGTGTTAGCAATGGTAGCGGTGGTAGTGGTAATGTTACCGGTTATGATACCTATGCAGCCGGTGCAGATAAATATCGTGGTTCTGGTGGCAGCGGTGGCCAGCTGACCACAGATGATGCAGAAAATAACACCGCAGTTGATTTAGGTAGTGGTGGTTCAGGTGGTAAAGAAGGTGATACCTTTGATTATAAAGGCGTGACCTTTACCATTGGTAGTGGCGGTAGCGGTACATTTACAATTGATACCAAAACCGGTGATGATGGCAATGGCAAAGTTGCAGCATATACCTTCGACTACAAAGGTGTTACGTTTACGATTGCAGCCTATGTGTATACCAGCGTTGTTAATGGTCAAGGTTCAGGCGGAAGTGGTGGTATTGTTGCAGAACGTCCGCAGACACCGAGCGCAAGCGAACATGTTGGCTCAGGTGGCAGTGGTCAGCAGACCCTGCAGAGCACCCCTCCGATTAGCGAAGAAAATAATGATGGCTCAGGCGGTTCAGGTCCGATGGCACAGCCTCGTACCACCATTAGTGATGCCGAAATTTGGGATGGCTCCGGTGGTAGCGGCAAAATGGTTCAGGATATTGATGGTCTGGGTGCACCGGGTAAAGATAGCGGCTCTGGCGGATCAGGCGCACCTGGCAAAGATAGTAAACTGGAAATGGATAACGCCAAATATCAGGCAGCCTATAAATACCAGGCATGGCAGAGCGGTTTTTAA。

[0066] SEQ ID NO.2:

[0067] MGSSHHHHHHSSGLVPRGSHMQDEIQQRLEEIDRVSNGSGGSGNVTGYDTYAAGADKYRGSGGSGGQLTTDDAENNTAVDLGSGGSGGKEGDTFDYKGVTFTIGSGGSGTFTIDTKTGDDGNGKVAAYTFDYKGVTF TIAAYVYTSVVNGQGSGGSGGIVAERPQTPSASEHVGSGGSGQQTLQSTPPISEENNDGSGGSGPMAQPRTTISDAEIWDGSGGSGKMVQDIDGLGAPGKDSGSGGSGAPGKDSKLEMDNAKYQAAYKYQAWQSGF*.

[0068] Example 1: Design of a multi-epitope subunit vaccine for Salmonella enteritidis

[0069] (1) Fasta sequence retrieval of protein whole genome

[0070] Suitable candidate antigen proteins in Salmonella enteritidis were screened, namely Salmonella enteritidis flagellar protein FliC and type III secretion system protein SipD; the FASTA sequences of Salmonella enteritidis FliC (KKE15205.1) and SipD (KKE05642.1) proteins were downloaded from NCBI.

[0071] (2) Prediction of protein immunogenicity

[0072] Immunogenicity analysis of the protein was performed using VaxiJen software (http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.htmL). "TARGET ORGANISM" was set to "Bacteria," and "Submit" was clicked. The antigenicity results were then viewed, with a threshold of 0.4; values ​​greater than 0.4 indicated a probable antigen.

[0073] (3) Protein signal peptide prediction

[0074] SignalP software (https: / / novopro.cn / tools / signalp.htmL) was used to predict the signal peptide of the protein. The protein species was selected as "Gram-negative bacteria", and possible signal peptide regions were eliminated based on the prediction results.

[0075] (4) Prediction of protein hydrophilicity

[0076] Input the protein sequence into the Protscale software (https: / / web.expasy.org / cgi-bin / protscale / protscale.pl?1). Based on the results, use 0 as the dividing line: >0 is a hydrophobic fragment, and <0 is a hydrophilic fragment. Fragments with high hydrophobicity values ​​are discarded.

[0077] (5) Prediction of protein transmembrane structure

[0078] Input the Fasta protein sequence into the TMHMM software (https: / / dtu.biolib.com / DeepTMHMM), click "Run", and observe whether a transmembrane structure exists.

[0079] (6) Prediction of protein toxicity and sensitization

[0080] To identify virulence proteins, BLASTP analysis of the protein sequences of virulence proteins in the Virulence Factors of Pathogenic Bacteria (VFDB) database was performed. Proteins meeting the following cutoff criteria were selected: position score >100, E value 1e-04. AllergenFPv.1.0 software was used to evaluate the sensitization potential of the proteins.

[0081] (7) Prediction of B-cell dominant epitopes and T-cell dominant epitopes

[0082] The B-cell dominant epitopes of the protein were predicted using ABCpred software (https: / / webs.iiitd.edu.in / raghava / abcpred / ABC_submission.html), with a threshold set to 0.51. Optimization was performed using BepiPred software. T-cell dominant epitopes of the protein were screened using EpiJen software (http: / / www.ddg-pharmfac.net / epijen / EpiJen / EpiJen.htm). The results are shown in Table 1.

[0083] B cell epitopes and antigenic epitopes that can bind to MHC class I and MHC class II molecules were predicted and screened from FliC and SipD proteins to obtain the recombinant protein FLPD (amino acid sequence as shown in SEQ ID NO.2), and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.1.

[0084] Table 1. Screening of B and T cell dominant epitopes for FliC and SipD proteins.

[0085]

[0086]

[0087] Note: The bolded sequences represent the selected antigenic epitopes.

[0088] (8) Prediction of protein physicochemical properties and structure

[0089] The Expasy ProtParam server was used to predict various physicochemical properties of the protein. The secondary structure of the obtained FLPD protein was evaluated using the online software SOPMA (https: / / npsa-pbil.ibcp.fr / cgi-bin / secpred_sopma.pl).

[0090] (9) Prediction of protein tertiary structure

[0091] Input the FLPD protein amino acid sequence into the I-TASSER software (https: / / zhanggroup.org / I-TASSER / ), click "Run I-TASSER," and obtain the 3D structure prediction map of the protein. The I-TASSER server is used to model the tertiary structure of the vaccine construct. The PyMOL program is used to visualize the generated protein's tertiary structure, such as... Figure 3 As shown.

[0092] (10) Vaccine molecular docking

[0093] The 3D structure of the multi-epitope protein FLPD was docked with immune receptor molecules MHC-I (PDB ID: 1L1Y), MHC-II (PDB ID: 1KG0), and TLR-4 (PDB ID: 4G8A) using the online analysis server Cluspro 2.0 (https: / / cluspro.bu.edu / login.php). The 3D structure of the docking complex with the lowest binding energy fraction was then visualized using PyMOL software. Figure 3 As shown.

[0094] (11) Electronic Immunization Simulation of Vaccines

[0095] The protein was simulated electronically using C-IMMSIM software (https: / / kraken.iac.rm.cnr.it / C-IMMSIM / index.php?page=1) to preliminarily predict its immunogenicity (Rapin et al 2010). Injections were administered at one-week intervals according to a typical immunization protocol, for a total of three immunizations. All simulation parameters were set to their default values, with time steps of 1, 42, and 63 (each time step being 8 hours, with time step 1 representing the first immunization).

[0096] Example 2: Induction and Purification of Protein

[0097] (1) Plasmid transformation and strain identification

[0098] The target gene (SEQ ID NO.1) was synthesized and constructed by Qingke Biotechnology Co., Ltd., using the recombinant plasmid pET-28b(+)-FLPD( Figure 2 The plasmid pET28b(+)-FLPD was transformed into *E. coli* BL21(DE3) competent cells and plated on LB / K an agar plates to obtain the recombinant expression strain, named *Escherichia coli* FLPD. Sequencing identification was performed, with primer sequences of: F: 5'-catatgcaggatgaaattcagcag-3' (SEQ ID NO.3), R: 5'-GAATTCTTA AAAACCGCTCTGCC-3' (SEQ ID NO.4). Sequencing alignment results are shown below. Figure 4 As shown.

[0099] The recombinant expression strain Escherichia coli FLPD was deposited at the China Center for Type Culture Collection (CCTCC) on January 17, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2024132.

[0100] (2) Induced expression of protein

[0101] IPTG was used to explore the induction conditions (concentration and time) for protein expression and to verify soluble expression. The induced multi-epitope proteins were analyzed by SDS-PAGE, and the results are shown below. Figure 5 The results showed that a 35.0 kDa FLPD protein was successfully expressed at an IPTG concentration of 1.0 mmol / L, a temperature of 37℃, and an induction time of 4 h, with the highest expression level. After ultrasonic disruption, the supernatant and precipitate were collected, revealing that the target protein was expressed in both the supernatant and precipitate, with a higher expression level in the supernatant, suitable for subsequent purification.

[0102] (3) Purification of multi-epitope proteins

[0103] ① Equilibrate the packed Ni NTABeads 6FF gravity column with 5 column volumes of Lysis Buffer to ensure the packing material is in the same buffer system as the target protein. Repeat 3 times.

[0104] ② Add the sample to the balanced gravity column and retain the sample for at least 2 minutes to ensure full contact between the sample and the medium. Collect the effluent.

[0105] ③ Use 10 times the volume of Wash Buffer to wash away non-specifically adsorbed proteins and collect the wash solution.

[0106] ④ Elute with 5 column volumes of Elution Buffer. Collect the remaining flow-through buffer, washing buffer, and elution buffer in fractions, one tube per column volume, and analyze them separately. This ensures that all bound target proteins are eluted and yields high-purity and high-concentration protein.

[0107] (4) Validation of multi-epitope proteins

[0108] ① The purified protein was concentrated by ultrafiltration and then verified by SDS-PAGE. The results are shown in the figure. Figure 6 The results showed that a highly purified multi-epitope protein was obtained after purification.

[0109] ② After performing SDS-PAGE on the multi-epitope proteins, they were validated using Western blot (the primary antibody used was a His-tag monoclonal antibody, and the secondary antibody was an HRP-labeled goat anti-mouse antibody). Figure 7 As shown.

[0110] (5) Removal of protein endotoxins

[0111] Because endotoxins from protein vaccines can cause fever in animals and interfere with the immune response by activating nonspecific immune responses, an endotoxin removal kit was used to remove the endotoxins. After endotoxin removal, the final endotoxin concentration in the protein was measured.

[0112] (6) Detection of multiepitope protein concentration

[0113] The protein concentration after ultrafiltration concentration was measured using a BCA protein concentration assay kit. The results showed that the protein concentration was 1.8 mg / mL.

[0114] (7) Detection of purity of multiepitope proteins

[0115] ① BSA standard protein at concentrations of 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL was analyzed by SDS-PAGE with the target protein (concentration A) of the desired purity. The results are shown in the figure. Figure 8 A.

[0116] ② The grayscale values ​​of the photomasks at different concentrations were measured using Image-J software. The results are shown in [Figure number missing]. Figure 8 B.

[0117] ③ The concentration B of the target protein can be calculated using the "concentration-grayscale value" standard curve.

[0118] ④ The B / A ratio represents the protein purity. The results are shown in Table 2.

[0119] Table 2 Protein purity data

[0120]

[0121] Example 3: Evaluation of the immunoprotective effect of Salmonella enteritidis multi-epitope subunit vaccine on mice.

[0122] (1) Mouse immunization procedure and antigen emulsification

[0123] Following the immunization schedule shown in Table 3, 20 healthy female BALB / c mice at 6 weeks of age were subcutaneously injected at multiple sites on the back of the neck. Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for booster immunizations. FLPD Salmonella multi-epitope vaccine was prepared by emulsifying 100 μL each of FLPD protein and adjuvant in equal proportions and then injected. Antigen emulsification: After diluting the immunogen with physiological saline, an equal volume of protein was added to either Freund's complete or incomplete adjuvant and emulsified thoroughly through a three-way valve. Emulsification test: A drop of the emulsified Freund's complete or incomplete adjuvant was dropped into cold water. If the droplet remained intact and floated on the surface, the emulsification was complete and the vaccine was ready for injection.

[0124] Table 3. Mouse Immunization Schedule

[0125]

[0126] (2) Immunogen safety identification

[0127] Following the immunization schedule in Table 3, FLPD protein was subcutaneously injected into BALB / c mice at multiple sites at doses of 20 μg / mouse, 50 μg / mouse, and 90 μg / mouse, respectively. During the immunization period, we observed and recorded adverse reactions and weight changes in the mice. No adverse reactions were observed in the immunized mice; their feed intake, feces, fur, and mental state were all normal. Regarding weight gain during the immunization period, there was no significant difference in weight gain among the different groups. Figure 9 As shown above, this immunogen does not exhibit toxicity in mice at normal doses, indicating that it has good safety.

[0128] (3) ELISA detection of specific antibody titers and cytokine levels in mouse serum

[0129] Serum was collected from the tail vein of mice after the first, second, and third immunizations, and the specific IgG titer was detected by indirect ELISA. The results are as follows: Figure 10As shown. The levels of the cytokines interferon-γ (IFN-γ), interleukin 4 (IL-4), interleukin 6 (IL-6), and interleukin 12 (IL-12) in mouse serum were measured using a double-antibody sandwich ELISA method. The results are shown below. Figure 11 As shown. For specific methods, please refer to the kit instructions (Mouse IL-6, Mouse IL-4, Mouse IFN-γ, Mouse IL-12p70, Hangzhou Lianke Biotechnology Co., Ltd.).

[0130] (4) Salmonella LD50 in mice 50 Measurement

[0131] Determination of the LD50 of Salmonella enteritidis standard strain CMCC(B)50335 and Salmonella typhimurium standard strain CVCC541 in BALB / c mice 50 Sixty female, 6-week-old SPF-grade BALB / c mice were randomly divided into 12 experimental groups, with 5 mice in each group. Each bacterial strain was further divided into 6 dosage groups. The bacterial suspensions were adjusted to a 0.5 McFarland turbidity standard, containing approximately 1.0 × 10⁻⁶ bacteria. 8 CFU / mL, six concentration gradients of Salmonella enteritidis were 3.0 × 10⁻⁶ CFU / mL. 7 CFU / mL, 3.0×10 6 CFU / mL, 3.0×10 5 CFU / mL, 3.0×10 4 CFU / mL, 3.0×10 3 CFU / mL and 3.0×10 2 CFU / mL; the six concentration gradients of Salmonella Typhimurium were 2.4 × 10⁻⁶. 7 CFU / mL, 2.4×10 6 CFU / mL, 2.4×10 5 CFU / mL, 2.4×10 4 CFU / mL, 2.4×10 3 CFU / mL and 2.4×10 2 CFU / mL. Mice in each group were sequentially injected intraperitoneally with different concentrations of Salmonella bacterial solution, 200 μL per mouse. After injection, the mice were housed separately and observed for 14 consecutive days. Morbidity and mortality were recorded.

[0132] Based on the experimental results, the modified Koch method was used to calculate the median lethal dose (LD50). 50 The calculation formula for LD is as follows: 50 =lg-1[Xm-i(∑P-0.5)];Sx 50=i[(∑P-∑P2) / (n-1)]1 / 2; LD 50 95% confidence limit = lg-1(1g LD) 50 ±1.96Sx 50 In the formula: i is the group interval, i.e., the difference between the logarithmic doses of two adjacent groups; Xm is the maximum logarithmic dose value; P is the mouse mortality rate, expressed as a decimal; ∑P is the sum of the mortality rates of all groups of animals; Sx 50 This is the standard error. The final determination of the LD50 of Salmonella enteritidis strain CMCC(B)50335 in 6-week-old BALB / c mice was performed. 50 1.20×10 4 CFU, Standard Error Sx 50 It is 0.37 CFU, LD 50 The 95% confidence limit is 2.24 × 10⁻⁶. 3 CFU ~ 6.46 × 10 4 CFU. Modified Kohl's method for determining the LD50 of Salmonella Typhimurium strain CVCC541 in 6-week-old BALB / c mice. 50 It is 6.03×10 2 CFU, Standard Error Sx 50 It is 0.37 CFU, LD 50 The 95% confidence limit is 1.12 × 10⁻⁶. 2 CFU ~ 3.24 × 10 3 CFU. The specific mortality rate of the mice is shown in Table 4.

[0133] Table 4. Results of Median Lethal Dose in Mice

[0134]

[0135] (5) Survival curves of mice after challenge

[0136] BALB / c mice were intraperitoneally injected with 10 LD50 5 days after the first booster immunization and 5 days after the second booster immunization. 50 Salmonella enteritidis standard strain CMCC(B)50335 and 10LD 50 Salmonella Typhimurium standard strain CVCC541 was injected into each mouse at a dose of 200 μL. A negative control was injected with an equal volume of 200 μL PBS. Mice were observed for 14 days post-challenge, and their survival status and mortality were recorded. Survival curves were plotted, as shown below. Figure 12 As shown.

[0137] (6) Detection of bacterial load in mouse liver and spleen

[0138] To evaluate the protective effect of the FLPD vaccine against Salmonella-infected organs in mice, the bacterial load in the spleen and liver of mice was measured 3 days after challenge. The results are as follows: Figure 13As shown.

[0139] Example 4: Evaluation of the immunoprotective effect of Salmonella enteritidis multi-epitope subunit vaccine on chicks

[0140] Immunization and challenge protection tests were conducted on SPF White Leghorn chicks using the FLPD Salmonella multiepitope vaccine (prepared as in Example 3). A commercially available attenuated live Salmonella enteritidis vaccine (Sm24 / Rif12 / Ssq strain) was used as a positive control. A series of tests, including chick weight changes, specific antibody levels, and protection rates, were conducted to evaluate the immunoprotective efficacy of the FLPD multiepitope vaccine against Salmonella enteritidis-infected chicks and to preliminarily explore the cross-protective effect of FLPD against other Salmonella serotypes. Immunization schedule: Ten healthy 2-day-old male White Leghorn chicks were injected subcutaneously at multiple sites on the back of the neck. The initial immunization used a 1:1 mixture of antigen and Freund's complete adjuvant. Booster immunizations were performed on days 16 and 23, using a 1:1 mixture of antigen and Freund's incomplete adjuvant. The immunization dose was 100 μg / chick. Ten healthy male white Leghorn chicks aged 2 days were immunized with a commercial vaccine via drinking water, for a total of one immunization; another 15 healthy male white Leghorn chicks aged 2 days were not immunized and served as a negative control.

[0141] (1) LD50 of Salmonella enteritidis in chicks 50 Measurement

[0142] To determine the appropriate challenge dose for chicks, 28-day-old White Leghorn chicks were tested for Salmonella enteritidis LD50. 50 The mortality rate of chicks was determined using the modified Kohl's method. The LD50 of *Salmonella enteritidis* strains against 28-day-old White Leghorn chicks was also determined. 50 It is 6.31×10 10 CFU, Standard Error Sx 50 0.40, LD 50 The 95% confidence limit is 1.04 × 10⁻⁶. 10 CFU ~ 3.84 × 10 11 CFU.

[0143] Table 5 Results of Median Lethal Dose in Chicks

[0144]

[0145] (2) Changes in chick weight

[0146] To investigate the safety and protective effect of the FLPD vaccine on chicks, the weight gain and living conditions of unvaccinated chicks, FLPD-vaccinated chicks, chicks vaccinated with a commercial vaccine, and unvaccinated and unchallenged chicks were recorded from the initial immunization. Weight gain is shown in the table below. Figure 14 As shown.

[0147] (3) Detection of chick-specific IgG

[0148] Blood and serum were collected from the hearts of SPF chicks one day before the second vaccination, one day before the third vaccination, and three days after the third vaccination. The levels of specific IgG antibodies in the serum of chicks immunized with FLPD protein were measured using an indirect ELISA method. The results are as follows: Figure 15 As shown.

[0149] (4) Survival curve of chicks after viral challenge

[0150] Survival curves of chicks after viral infection are shown below. Figure 16 When using the absolute lethal dose (6.31 × 10⁻⁶), 11 After challenging chicks with CFU (Chronic Fluoropropyl Fibrosum) virus, most unvaccinated chicks exhibited lethargy, rough feathers, and huddling together, and subsequently died. Only a small percentage of vaccinated chicks showed these symptoms. After the immunization program was completed, challenge with Salmonella enteritidis resulted in a 100% mortality rate (10 / 10) for unvaccinated chicks two weeks later, while the mortality rate for vaccinated chicks with FLPD (Fluoropropyl Fibrosum) was 30% (3 / 10), with an immunization protection rate of 70%. Chicks vaccinated with commercially available vaccines had a mortality rate of 20% (2 / 10), with an immunization protection rate of 80%.

[0151] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A Salmonella multiepitope protein, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding a Salmonella multiepitope protein as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

3. A recombinant expression vector, characterized in that, Includes the coding gene as described in claim 2.

4. A recombinant host cell, characterized in that, Includes the recombinant expression vector as described in claim 3.

5. The recombinant host cell according to claim 4, characterized in that, The recombinant host cell was Escherichia coli FLPD; The *Escherichia coli* FLPD strain was deposited on January 17, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2024132.

6. The use of the encoding gene as described in claim 2, the recombinant expression vector as described in claim 3, or the recombinant host cell as described in claim 4 or 5 in the preparation of the Salmonella multiepitope protein as described in claim 1.

7. The application of the Salmonella multi-epitope protein as described in claim 1 in the preparation of Salmonella subunit vaccines, characterized in that, The Salmonella mentioned is Salmonella enteritidis and / or Salmonella typhimurium.

8. A Salmonella subunit vaccine, characterized in that, The active ingredient includes the Salmonella multiepitope protein as described in claim 1.

9. The Salmonella subunit vaccine according to claim 8, characterized in that, The Salmonella subunit vaccine also includes a vaccine adjuvant.

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