Salmonella multi-epitope protein, coding gene and application of salmonella multi-epitope protein in preparation of salmonella subunit vaccine

Abstract

The invention 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 technical field of biology. The amino acid sequence of the salmonella multi-epitope protein is as shown in SEQ ID NO. 2. The nucleotide sequence of the coding gene of the salmonella multi-epitope protein is as shown in SEQ ID NO. 1. The salmonella subunit vaccine prepared by utilizing the salmonella multi-epitope protein has the advantages of safety, stability, low cost, no toxicity to the body, capability of simultaneously causing the body to generate body fluid and cellular immunity and the like, not only can have a prevention and protection effect on salmonella enteritidis infection, but also can have a cross protection effect on salmonella typhimurium infection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Salmonella multi-epitope protein, a coding gene and an application thereof in preparing a Salmonella subunit vaccine. Background Art

[0002] Salmonella enteritidis is a facultative intracellular pathogen that can be carried asymptomatically in the intestines or gallbladders of livestock and poultry and can cause gastroenteritis. It is one of the main causes of foodborne diseases in humans worldwide. Antibiotic treatment is the main means to combat Salmonella enteritidis infection. However, given the increasing incidence of multidrug-resistant Salmonella in animals and the hazards of drug residues, the development of safe and effective Salmonella vaccines has received increasing attention.

[0003] The Salmonella vaccines currently under research mainly include inactivated vaccines, live attenuated vaccines and subunit vaccines. Inactivated vaccines do not induce cellular immunity and have poor immune effects; live attenuated vaccines are the most popular because they can stimulate the host's cellular and humoral immunity and provide long-term protective immune responses to the body, but there is a possibility of virulence recovery and it is inconvenient to distinguish between infected animals and immune animals; compared with whole-bacterial vaccines, subunit vaccines are safer, but often need to be used with adjuvants to improve the immune effect. On the other hand, the Salmonella genome is variable, so conventional subunit vaccines based on virulence factors may have limited applicability and cannot protect against heterovirulent strains, requiring new vaccine development strategies.

[0004] Multi-epitope vaccines use minimal immunogenic fragments to trigger effective immune responses against specific pathogens. Compared with entire homologous proteins, epitopes can induce immune responses more directly. While inducing humoral immunity and cellular immunity, they can overcome the limitations of traditional live attenuated vaccines, and have the advantages of good safety, low cost, ease of use, and easy distinction between infected animals and immune animals. In addition, proteins expressed by pathogens at different times and environments can be used as candidate antigens and combined for multi-epitope vaccines to improve the universal applicability of vaccines. Based on this, the present invention intends to develop a new multi-epitope subunit vaccine for Salmonella enteritidis, thereby providing effective immune protection against Salmonella infection. Summary of the invention

[0005] The purpose of the present invention is to provide a Salmonella multi-epitope protein, a coding gene and the use thereof in the preparation of a Salmonella subunit vaccine to solve the problems existing in the above-mentioned prior art. The Salmonella subunit vaccine prepared using the Salmonella multi-epitope protein has the advantages of safety, stability, low cost, non-toxicity to the body, and the ability to induce humoral and cellular immunity in the body at the same time, and can not only prevent and protect against Salmonella enteritidis infection, but also can play a cross-protective role against Salmonella typhimurium infection.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a Salmonella multi-epitope 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 multi-epitope protein, and the nucleotide sequence is shown as SEQ ID NO.1.

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

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

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

[0012] The Escherichia coli FLPD was deposited in the China Center for Type Culture Collection on January 17, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2024132.

[0013] The present invention also provides the use of the above-mentioned encoding gene, recombinant expression vector or recombinant host cell in preparing Salmonella multi-epitope protein.

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

[0015] Furthermore, the Salmonella is Salmonella Enteritidis and / or Salmonella Typhimurium.

[0016] The present invention also provides a Salmonella subunit vaccine, the active ingredients of which include the above-mentioned Salmonella multi-epitope protein.

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

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

[0019] The present invention predicts and screens B cell epitopes and antigenic epitopes that can bind to MHC-I and MHC-II molecules from FliC and SipD proteins, prepares a Salmonella multi-epitope protein FLPD through linker fusion expression, and prepares a Salmonella subunit vaccine using the Salmonella multi-epitope protein FLPD. The Salmonella subunit vaccine has the advantages of safety, stability, low cost, non-toxicity to the body, and the ability to simultaneously induce humoral and cellular immunity in the body. It can not only prevent and protect against Salmonella enteritidis infection, but also cross-protect against Salmonella typhimurium infection. In addition, this method does not require the cultivation of microorganisms and can also play an effective protective role against some mutant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It 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 tertiary structure of FLPD protein and the molecular docking results; A is the tertiary structure of protein FLPD; B is the docking model of FLPD protein and MHC-I molecule; C is the docking model of FLPD protein and MHC-II molecule; D is the docking model of FLPD protein and TLR-4 receptor molecule;

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

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

[0026] Figure 6Figure 1 is the result of SDS-PAGE and Western-blot detection after FLPD protein purification; A is the result of protein purification SDS-PAGE: Lane M is the protein marker; Lanes 1-6 are the whole bacteria before high-pressure disruption, the whole bacteria after high-pressure disruption, the supernatant, the flow-through, the flow-through, and the precipitate; Lanes 7-8 are the washing solution; Lanes 9-10 are the 50mmol / L imidazole eluent; Lanes 11-13 are the 100mmol / L imidazole eluent; Lane 14 is the 2 00mmol / L imidazole eluent; lanes 15-18 are 200mmol / L imidazole eluent; lanes 19-22 are 250mmol / L imidazole eluent; lane 23 is 300mmol / L imidazole eluent; lane 24 is 500mmol / L imidazole eluent; 25-28 are whole bacteria, supernatant, supernatant, and precipitate after high-pressure crushing; B is protein Western-blot verification: lane M is protein marker; lane 1 is the purified protein FLPD;

[0027] Figure 7 It is a graph of protein purity test results; wherein, lane M is protein marker; lanes 1-7 are BSA standard proteins with concentrations of 0.5mg / mL, 0.4mg / mL, 0.3mg / mL, 0.2mg / mL, 0.1mg / mL, 0.05mg / mL, and 0.025mg / mL, respectively; lane 8 is 250mmol / L imidazole eluent; lane 9 is 200mmol / L, 250mmol / L imidazole fusion tube; lane 10 is 300mmol / L imidazole eluent; lane 11 is 200mmol / L imidazole eluent; lane 12 is 200mmol / L imidazole eluent; lane 13 is 250mmol / L imidazole eluent; lane 14 is 250mmol / L imidazole eluent;

[0028] Figure 8 The standard curves of protein concentration and A562 (A) and protein concentration and gray value (B);

[0029] Fig. 9 The figure is a statistical chart of weight changes of mice immunized with different doses;

[0030] Fig.10 This is a statistical chart of specific IgG antibody titer in the serum of immunized mice;

[0031] Fig.11 Statistical graphs of the expression levels of mouse serum cytokines IFN-γ (A), IL-4 (B), IL-6 (C), and IL-12 (D);

[0032] Fig.12The following are the survival curves of mice after virus attack; A is the survival curve of Enteritidis virus attack after two immunizations; B is the survival curve of Salmonella typhimurium virus attack after two immunizations; C is the survival curve of Enteritidis virus attack after three immunizations; D is the survival curve of Salmonella typhimurium virus attack after three immunizations;

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

[0034] Fig.14 This is a statistical chart of chick weight changes;

[0035] Fig.15 This is a statistical chart of specific IgG antibody titer in sera of immunized chickens;

[0036] Fig.16 This is the chick survival curve. DETAILED DESCRIPTION

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

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

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

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

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

[0045] 3) The antigenicity, allergenicity, hydrophilicity, signal peptide region and transmembrane structure of the candidate antigens FliC and SipD were predicted to ensure that the selected proteins were non-toxic, non-allergenic, hydrophilic, and free of signal peptide regions and transmembrane structures.

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

[0047] 5) The antigen epitope sequence obtained by screening is expressed through linker fusion to prepare the recombinant protein FLPD.

[0048] 6) The recombinant protein FLPD is mixed with an adjuvant in equal proportions at a certain concentration to obtain a Salmonella multi-epitope subunit vaccine.

[0049] 7) The prepared FLPD Salmonella multi-epitope vaccine was used to carry out immunization and challenge protection experiments on SPF BALB / c mice, and the protective effect of the FLPD vaccine was evaluated through a series of experiments including vaccine safety, specific antibody titer, cytokine levels, survival curves, organ bacterial loads, and tissue pathology evaluation.

[0050] 8) The prepared FLPD Salmonella multi-epitope vaccine was used to carry out immunization and challenge protection tests on SPF white Leghorn chicks, and a commercial attenuated live vaccine of Enteritidis Salmonella (Sm24 / Rif12 / Ssq strain) was used as a positive control to immunize chicks. A series of tests such as chick weight changes, specific antibody levels and protection rates were conducted to evaluate the immune protective efficacy of the multi-epitope vaccine FLPD on chicks infected with Enteritidis Salmonella, and to preliminarily explore the cross-protection effect of FLPD against other Salmonella serotypes.

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

[0052] 220 SPF 6-week-old BALB / c mice were provided by the Experimental Animal Center of Huazhong Agricultural University. The first batch of 60 mice was used to determine the median lethal dose (LD) of Salmonella enteritidis and Salmonella typhimurium in mice. 50 ) experiment. The second batch of 160 mice was used for vaccine immunity research. They were raised in the Experimental Animal Center of Huazhong Agricultural University. Ethics number: HZAUMO-2024-0030.

[0053] Two batches of SPF white Leghorn chicks: the first batch of 3-week-old chicks and the second batch of 1-day-old chicks were purchased from Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd. The first batch of SPF chicks, 20 chicks, were used for the LD test of Salmonella Enteritidis in chicks. 50 Experiment. The second batch of 35 SPF chicks were used for vaccine immunization research. They were raised in a stainless steel poultry isolator at the Department of Veterinary Science of Huazhong Agricultural University, with free access to water and SPF chick feed. The temperature was controlled at 36℃~37℃ in the first week, and then decreased by 1℃ every day until it dropped to 30℃. The room temperature was controlled at 24℃. Ethics number: HZAUCH-2024-0005.

[0054] The experimental research of the present invention was carried out in accordance with the guidelines of the "Regulations on the Administration of Laboratory Animals in China" (1988) and the "Regulations on the Administration of Laboratory Animals in Hubei Province" (2005). In the present invention, all work was done to treat the experimental animals in an ethical manner and to minimize the suffering of the experimental animals.

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

[0056] 2% agarose gel: Weigh 1.2 g of agarose, heat and dissolve in 60 mL of 1×TAE buffer, cool to about 50°C, add nucleic acid dye, mix well, pour into the gel plate, and use after it is fully solidified.

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

[0058] IPTG (isopropylthio-β-D-galactoside): Dissolve 2 g IPTG in 8 mL distilled water, dilute to 10 mL with distilled water, filter and sterilize with a 0.22 μm filter, divide into 1 mL portions and store at -20°C. The prepared IPTG concentration is 0.8 mmol / 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 in 900 mL of double distilled water, add double distilled water to make up to 1 L, adjust the pH to 8.0, filter with a 0.22 μm aqueous 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 in 900 mL of double distilled water, add double distilled water to make up to 1 L, adjust the pH to 8.0, filter with a 0.22 μm aqueous 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 in 900 mL of double distilled water, add double distilled water to make up to 1 L, adjust the pH to 8.0, filter with a 0.22 μm aqueous filter membrane, and store at 4°C.

[0062] Coomassie Brilliant Blue staining solution: Accurately weigh 1g of Coomassie Brilliant Blue R-250, place it in a 1L beaker, measure and add 250mL of isopropanol, 100mL of glacial acetic acid and 650mL of deionized water, stir evenly, 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 use.

[0064] SEQ ID NO.1:

[0065] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCAGGATGAAATTCAGCAGCGTCTGGAAGAAATTGATCGTGTTAGCAATGGTAGCGGTGGTAGTGGTAATGTTACCGGTTATGATACCTATGCAGCCGGTGCAGATAAATATCGTGGTTCTGGTGGCAGCGGTGGCCAGCTGACCACAGATGATGCAGAAAATAACACCGCAGTTGATTTAGGTAGTGGTGGTTCAGGTGGTAAAGAAGGTGATACCTTTGATTATAAAGGCGTGACCTTTACCATTGGTAGTGGCGGTAGCGGTACATTTACAATTGATACCAAAACCGGTGATGATGGCAATGGCAAAGTTGCAGCATATACCTTCGACTACAAAGGTGTTACGTTTACGATTGCAGCCTATGTGTATACCAGCGTTGTTAATGGTCAAGGTTCAGGCGGAAGTGGTGGTATTGTTGCAGAACGTCCGCAGACACCGAGCGCAAGCGAACATGTTGGCTCAGGTGGCAGTGGTCAGCAGACCCTGCAGAGCACCCCTCCGATTAGCGAAGAAAATAATGATGGCTCAGGCGGTTCAGGTCCGATGGCACAGCCTCGTACCACCATTAGTGATGCCGAAATTTGGGATGGCTCCGGTGGTAGCGGCAAAATGGTTCAGGATATTGATGGTCTGGGTGCACCGGGTAAAGATAGCGGCTCTGGCGGATCAGGCGCACCTGGCAAAGATAGTAAACTGGAAATGGATAACGCCAAATATCAGGCAGCCTATAAATACCAGGCATGGCAGAGCGGTTTTTAA。

[0066] SEQ ID NO.2:

[0067] MGSSHHHHHHSSGLVPRGSHMQDEIQQRLEEIDRVSNGSGGSGNVTGYDTYAAGADKYRGSGGSGGQLTTDDAENNTAVDLGSGGSGGKEGDTFDYKGVTFTIGSGGSGTFTIDTKTGDDGNGKVAAYTFDYKGVTF TIAAYVYTSVVNGQGSGGSGGIVAERPQTPSASEHVGSGGSGQQTLQSTPPISEENNDGSGGSGPMAQPRTTISDAEIWDGSGGSGKMVQDIDGLGAPGKDSGSGGSGAPGKDSKLEMDNAKYQAAYKYQAWQSGF*.

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

[0069] (1) Protein whole genome Fasta sequence retrieval

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

[0071] (2) Protein immunogenicity prediction

[0072] Use VaxiJen software (http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.htmL) to analyze the immunogenicity of the protein. Select "Bacteria" in "TARGET ORGANISM" and click "Submit" to view the antigenicity results. The threshold is 0.4, and values ​​greater than 0.4 are possible antigens.

[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, and the protein species was selected as "Gram-negative bacteria". Based on the prediction results, possible signal peptide regions were eliminated.

[0075] (4) Protein hydrophilicity prediction

[0076] The protein sequence was input into the Protscale (https: / / web.expasy.org / cgi-bin / protscale / protscale.pl?1) software, and according to the results, 0 was used as the dividing line, >0 was a hydrophobic fragment, <0 was a hydrophilic fragment, and fragments with high hydrophobic values ​​were eliminated.

[0077] (5) Protein transmembrane structure prediction

[0078] Input the protein Fasta sequence into the TMHMM software (https: / / dtu.biolib.com / DeepTMHMM), click “Run” to observe whether the result contains a transmembrane structure.

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

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

[0081] (7) Prediction of protein 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), and the Threshold was set to 0.51. The BepiPred software was used for optimization and selection. The T cell dominant epitopes of the protein were screened using EpiJ en (http: / / www.ddg-pharmfac.net / epijen / EpiJen / EpiJen.htm) software. The results are shown in Table 1.

[0083] B cell epitopes and antigen epitopes that can bind to MHC-I and MHC-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 table of B and T cell dominant epitopes of FliC protein and SipD protein

[0085]

[0086]

[0087] Note: The bold sequences represent the screened antigen epitopes.

[0088] (8) Protein physicochemical property structure prediction

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

[0090] (9) Protein tertiary structure prediction

[0091] Input the FLPD protein amino acid sequence into the I-TASSER software (https: / / zhanggroup.org / I-TASSER / ), click “Run I-TASSER” to get the 3D structure prediction diagram 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 tertiary structure of the generated protein, such as Figure 3 shown.

[0092] (10) Vaccine Molecular Docking

[0093] The 3D structure of the multi-epitope protein FLPD was docked with the immune receptor molecules MHC-I (PDB ID: 1L1Y), MHC-II (PDB ID: 1KG0) and TLR-4 (PDB ID: 4G8A) using the online analysis server Cluspro2.0 (https: / / cluspro.bu.edu / login.php), and the 3D structure of the docked complex with the lowest binding energy score was visualized and analyzed using the PyMOL software, as shown in Figure 2. Figure 3 shown.

[0094] (11) Vaccine electronic immune simulation

[0095] The protein was simulated electronically using C-IMMSIM (https: / / kraken.iac.rm.cnr.it / C-IMMSIM / index.php?page=1) software to preliminarily predict the immune efficacy of the protein (Rapin et al 2010). The typical immunization process was performed with injections every 1 week, for a total of 3 immunizations. All simulation parameters were set to default values, and the time step was set to 1, 42, and 63 (each time step was 8 hours, and time step 1 was the first immunization time point).

[0096] Example 2 Induced expression and purification of protein

[0097] (1) Plasmid transformation and strain identification

[0098] The target gene (SEQ ID NO.1) was synthesized by Qingke Biotechnology Co., Ltd. and the recombinant plasmid pET-28b(+)-FL PD( Figure 2 ). The plasmid pET28b(+)-FLPD was transformed into Escherichia coli BL21(DE3) competent cells and spread on LB / K an agar plates to obtain recombinant expression bacteria, named Escherichia coli FLPD. Sequencing was performed to identify the recombinant bacteria. The primer sequences were: F: 5'-catatgcaggatgaaattcagcag-3' (SEQ ID NO.3), R: 5'-GAATTCTTA AAAACCGCTCTGCC-3' (SEQ ID NO.4). The sequencing results are shown in Figure 4 shown.

[0099] The recombinant expression bacteria Escherichia coli FLPD was deposited in the China Center for Type Culture Collection on January 17, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024132.

[0100] (2) Inducible expression of proteins

[0101] IPTG was used to explore the induction expression conditions (concentration, time) and verify the soluble expression. The induced multi-epitope protein was tested by SDS-PAGE. The results are shown in Figure 5 The results showed that the FLPD protein with a size of 35.0 kDa was successfully expressed at an IPTG concentration of 1.0 mmol / L, a temperature of 37°C, and an induction time of 4 h, and the expression level was the highest. After ultrasonic disruption, the supernatant and precipitate were taken separately, and it was found that the target protein could be expressed in both the supernatant and the precipitate, and the expression level in the supernatant was high, which could be used 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 keep the filler in the same buffer system as the target protein. Repeat 3 times.

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

[0105] ③ Wash with 10 times the volume of Wash Buffer to remove non-specifically adsorbed impurities and collect the wash buffer.

[0106] ④ Use 5 times column volume of Elution Buffer for elution. Collect the remaining flow-through, washing solution and elution solution, collect them in sections, collect one tube for each column volume, and test them separately, which can ensure that all bound target proteins are eluted and obtain high-purity and high-concentration proteins.

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

[0108] ① After ultrafiltration and concentration of the purified protein, SDS-PAGE was performed for verification. The results are shown in Figure 6 The results showed that a multi-epitope protein with high purity was obtained after purification.

[0109] ②After SDS-PAGE, the multi-epitope protein was verified by Western blot (the primary antibody was His-tag monoclonal antibody, and the secondary antibody was HRP-labeled goat anti-mouse antibody). Figure 7 shown.

[0110] (5) Protein endotoxin removal

[0111] Since protein vaccine endotoxins may cause fever in animals and interfere with immune responses by activating nonspecific immune responses, endotoxin removal kits were used to remove endotoxins. Protein endotoxin detection was performed. After protein endotoxins were removed, the final endotoxin concentration was 0.095EU / mL.

[0112] (6) Detection of multi-epitope protein concentration

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

[0114] (7) Purity detection of multi-epitope proteins

[0115] ① Perform SDS-PAGE detection on BSA standard proteins with 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 and the target protein to be tested for purity (concentration A). The results are shown in Figure 8 Middle A.

[0116] ② The grayscale values ​​of different concentrations of the gel image were measured using Image-J software. The results are shown in Figure 8 Middle B.

[0117] ③The concentration B corresponding to the target protein can be calculated through the "concentration-gray value" standard curve.

[0118] ④The value of B / A is the protein purity. The results are shown in Table 2.

[0119] Table 2 Protein purity data

[0120]

[0121] Example 3 Evaluation of the immune protection effect of multi-epitope subunit vaccine of Salmonella enteritidis on mice

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

[0123] According to the immunization program shown in Table 3, 20 healthy female BALB / c mice aged 6 weeks were subcutaneously injected at multiple points on the back of the neck. Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for the booster immunization. FLPD protein and adjuvant 100 μL each were emulsified in equal proportions to obtain the FLPD Enteritidis Salmonella multi-epitope vaccine for injection. Antigen emulsification: After the immunogen was diluted with physiological saline, Freund's complete adjuvant or Freund's incomplete adjuvant was taken, and an equal volume of protein was added to fully emulsify and mix through a three-way valve. Emulsification identification: The above-mentioned emulsified and mixed Freund's complete adjuvant or Freund's incomplete adjuvant was dropped into cold water. The droplets remained intact and floated on the water surface, indicating that the emulsification was complete and could be injected.

[0124] Table 3 Mouse immunization schedule

[0125]

[0126] (2) Safety evaluation of immunogens

[0127] According to the immunization schedule in Table 3, FLPD protein was subcutaneously injected into BALB / c mice at multiple points at a dose of 20 μg / mouse, 50 μg / mouse, and 90 μg / mouse. During the immunization period, we observed and recorded the adverse reactions and weight changes of the mice. It was found that the immunized mice had no adverse reactions, and their feeding, feces, hair, and mental state were normal. According to the weight gain of mice during the immunization period, there was no significant difference in the weight gain of mice in different groups, such as Fig. 9 The above shows that the immunogen has no toxic reaction to mice at normal doses and has good safety.

[0128] (3) ELISA detection of mouse serum specific antibody titer and cytokine content

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

[0130] (4) LD of Salmonella in mice 50 Determination of

[0131] Determination of the LD of the standard strains of Salmonella Enteritidis CMCC(B)50335 and Salmonella Typhimurium CVCC541 in BALB / c mice 50 . 60 female, 6-week-old SPF BALB / c mice were randomly divided into 12 experimental groups, 5 mice in each group. Each strain was divided into 6 dose groups. The bacterial solution was adjusted to 0.5 McFarland turbidity standard, containing about 1.0×10 8 CFU / mL, the six concentration gradients of Enteritidis Salmonella were 3.0×10 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 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. Each group of mice was intraperitoneally injected with the above-mentioned Salmonella solutions of different concentrations, 200 μL per mouse. After injection, the mice were kept in separate cages and observed for 14 consecutive days. The incidence and mortality of the mice were recorded and recorded.

[0132] According to the test results, the modified Koch method was used to calculate the median lethal dose, LD 50 The calculation formula of 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 distance, that is, the difference between the logarithmic doses of two adjacent groups; Xm is the logarithmic value of the maximum dose; P is the mouse mortality rate, expressed as a decimal; ∑P is the sum of the mortality rates of animals in each group; Sx 50 The LD of Enteritidis strain CMCC(B)50335 against 6-week-old BALB / c mice was finally determined. 50 1.20×10 4 CFU, standard error Sx 50 0.37 CFU, LD 50 The 95% confidence limit is 2.24×10 3 CFU~6.46×10 4 CFU. Determination of LD of Salmonella typhimurium strain CVCC541 in 6-week-old BALB / c mice by modified Koch method 50 6.03×10 2 CFU, standard error Sx 50 0.37 CFU, LD 50 The 95% confidence limit is 1.12×10 2 CFU~3.24×10 3 CFU. The specific death conditions of mice are shown in Table 4.

[0133] Table 4 Results of median lethal dose in mice

[0134]

[0135] (5) Survival curve of mice after infection

[0136] BALB / c mice were injected intraperitoneally with 10LD 50 Salmonella enteritidis standard bacteria CMCC(B)50335 and 10LD 50 Each mouse was injected with 200 μL of the standard Salmonella typhimurium CVCC541. The negative control was injected with an equal amount of 200 μL PBS. The mice were observed for 14 days after the challenge, and the living conditions of the mice were observed and the deaths of the mice were recorded. The survival curve of the mice was drawn, as shown in Fig.12 shown.

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

[0138] To evaluate the protective effect of FLPD vaccine on the organs of mice infected with Salmonella, the bacterial load in the spleen and liver of mice was detected 3 days after infection. Fig.13shown.

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

[0140] The FLPD Salmonella multi-epitope vaccine (preparation method is the same as in Example 3) was used to carry out immunization and challenge protection tests on SPF white Leghorn chicks, and the commercial attenuated live vaccine of Enteritidis Salmonella (Sm24 / Rif12 / Ssq strain) was used as a positive control to immunize chicks. A series of tests such as the change in chick weight, the level of specific antibodies and the protection rate were performed to evaluate the immune protection efficacy of the multi-epitope vaccine FLPD on chicks infected with Enteritidis Salmonella, and the cross-protection effect of FLPD on other Salmonella serotypes was preliminarily explored. Immunization procedure: 10 healthy male white Leghorn chicks aged 2 days were injected subcutaneously at multiple points on the back of the neck, wherein the initial immunization used a 1:1 mixture of antigen + Freund's complete adjuvant, and booster immunizations were used on the 16th and 23rd days, respectively, and the booster immunizations used a 1:1 mixture of antigen + Freund's incomplete adjuvant. The immunization dose was 100 μg / feather. Ten 2-day-old healthy male white Leghorn chicks were immunized with commercial vaccines through drinking water, for a total of one immunization; another 15 2-day-old healthy male white Leghorn chicks were not immunized and served as negative controls.

[0141] (1) Effect of Salmonella enteritidis on LD in chicks 50 Determination

[0142] In order to determine the chick immune challenge dose, 28-day-old white Leghorn chicks were tested for Salmonella Enteritidis LD 50 The mortality of chicks is shown in Table 5. The LD of Enteritidis strains against 28-day-old white Leghorn chicks was determined by the modified Koch method. 50 6.31×10 10 CFU, standard error Sx 50 0.40, LD 50 The 95% confidence limit is 1.04×10 10 CFU~3.84×10 11 CFU.

[0143] Table 5 Results of median lethal dose for chicks

[0144]

[0145] (2) Changes in chick weight

[0146] In order to explore the safety and protective effect of the FLPD vaccine on chicks, the weight gain and living conditions of unimmunized chicks, FLPD-immunized chicks, commercial vaccine-immunized chicks, and unimmunized and non-challenged chicks were recorded from the first immunization. Fig.14 shown.

[0147] (3) Chick-specific IgG detection

[0148] Blood was collected from the hearts of SPF chickens one day before the second immunization, one day before the third immunization, and the third day after the third immunization, and serum was separated. The serum of chickens immunized with FLPD protein was tested for specific IgG antibody levels using indirect ELISA. The results are as follows: Fig.15 shown.

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

[0150] The survival curve of chicks after infection is shown in Fig.16 When the absolute lethal dose (6.31×10 11 After the chicks were challenged with FLPD (CFU), most of the non-immunized chicks showed signs of listlessness, ragged fur, and huddling, and died one after another, while only a small number of the immunized chicks showed the above-mentioned phenomena. After the immunization program was completed, it was found that the mortality rate of non-immunized chicks challenged with Salmonella enteritidis was 100% (10 / 10) two weeks later, and the mortality rate of FLPD-immunized chicks was 30% (3 / 10), and the immune protection rate was 70%. The mortality rate of chicks immunized with commercial vaccines was 20% (2 / 10), and the immune protection rate was 80%.

[0151] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A multi-epitope protein of Salmonella, It is characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding the multi-epitope protein of Salmonella as claimed in claim 1, It is characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

3. A recombinant expression vector, It is characterized in that Comprising the coding gene described in claim 2.

4. A recombinant host cell, It is characterized in that Comprising the recombinant expression vector according to claim 3.

5. The recombinant host cell according to claim 4, It is characterized in that The recombinant host cell is Escherichia coli FLPD; The Escherichia coli FLPD was deposited in the China Center for Type Culture Collection on January 17, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2024132.

6. Use of the encoding gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant host cell according to claim 4 or 5 in preparing the Salmonella multi-epitope protein according to claim 1.

7. Use of the Salmonella multi-epitope protein as claimed in claim 1 in the preparation of Salmonella subunit vaccine.

8. The use according to claim 7, It is characterized in that The salmonella is salmonella enteritidis and / or salmonella typhimurium.

9. A Salmonella subunit vaccine, It is characterized in that The active ingredient comprises the Salmonella multi-epitope protein according to claim 1.

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

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