Preparation method and application of a multi-epitope genetically engineered subunit vaccine against porcine Pasteurella multocida

Abstract

The present invention discloses a preparation method and application of a polyepitope genetically engineered subunit vaccine for porcine Pasteurella multocida, which belongs to the field of genetic engineering in biotechnology. The method comprises: selecting 6 important structural proteins of Pm by literature search, analyzing and screening the sequences of amino acids encoded by the genes of important structural proteins of Pm by bioinformatics analysis software, screening T cell antigen epitopes that can bind to MHC-I / II class molecules and B cell antigen epitopes of peptide or protein surface residues that can bind to antibodies from the screened virulence factors, constructing and synthesizing polyepitope fusion antigen genes and expressing them in Escherichia coli. The antigen epitopes are integrated into a recombinant protein PME and mixed with an adjuvant to obtain a polyepitope genetically engineered subunit vaccine for porcine Pasteurella multocida, which has the advantages of good safety, low cost, ease of use, and easy distinction between infected animals and immune animals, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering in biotechnology, and in particular relates to a preparation method and application of a multi-epitope genetically engineered subunit vaccine of porcine Pasteurella multocida, specifically involving the prediction, prokaryotic expression and purification of T cell and B cell antigen epitopes. Background Art

[0002] Pasteurellosis in pigs is an acute, epidemic, sporadic, and secondary infectious disease caused by Pasteurella multocida (Pm), characterized by fibrinous pleuropneumonia, pharyngitis, and septicemia. Affected pigs experience significant symptoms such as high fever, wheezing, difficulty breathing, and loss of appetite. The most acute infection often presents with clinical symptoms of septicemia, resulting in death within a day. The disease is highly contagious, highly harmful, rapidly spreads, and has a high mortality rate, severely impacting the growth and development of pigs on large-scale farms and hindering the development of pig farms and the pig farming industry. The disease often co-infects with other diseases, such as swine erysipelas and streptococcosis, further complicating the condition and making cure even more difficult. With the overuse of antibiotics, drug-resistant Pm strains have emerged in large numbers, with an increasingly broad spectrum of resistance, rendering conventional antibiotics ineffective. This makes immunoprevention a crucial approach to disease prevention and control.

[0003] Inactivated vaccines, prepared by inactivating bacteria through exogenous methods such as heating, drying, and formaldehyde and then mixing them with an adjuvant, are an effective means of preventing Pasteurellosis (PM). Generally, the inactivated vaccine strain is selected, and the locally dominant strain is isolated and cultured. Then, it is treated with physical and chemical methods to inactivate its pathogenicity while maintaining good immunogenicity. Finally, it is emulsified with an adjuvant, effectively reducing mortality in pigs. Inactivated vaccines offer poor cross-protection, a prolonged immune response, short-lived antibody retention, and require multiple immunizations. Attenuated live vaccines are prepared by artificially destroying antigenic components such as capsular polysaccharides from virulent PM strains through chemical and physical methods, causing them to be lost or mutated. The resulting attenuated strain is screened for. However, attenuated vaccines carry the risk of relapse, a significant risk. Therefore, the development of new vaccines that effectively prevent Pasteurellosis is a top priority.

[0004] A genetically engineered subunit vaccine is a vaccine made by purifying a protein antigen expressed through genetic engineering. A subunit vaccine is a vaccine that uses genetic engineering technology to connect the antigen gene of swine Pasteurella multocida to a vector for expression. It can significantly stimulate the host's humoral immunity and cellular immunity, and produce a high level of protective antibodies. The expression systems for exogenous antigens mainly include bacteria, yeast, mammalian cells, and insect cells. Genetically engineered subunit vaccines have good safety. Adjuvants are usually used to increase immunogenicity. They have a single component, no irrelevant antigens, and no problem of virulence reversion. Multi-epitope vaccines can achieve the goal of treating multiple serotypes with one vaccine, provide cross-immune protection, and reduce treatment costs. Multi-epitope vaccines have good application prospects due to their good safety, low cost, ease of use, and easy distinction between infected and immunized animals. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a multi-epitope genetically engineered subunit vaccine of porcine Pasteurella multocida and its application, so as to solve the problems existing in the above-mentioned prior art.

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

[0007] One of the technical solutions of the present invention is a recombinant protein PME, whose amino acid sequence is shown in SEQ ID NO.1.

[0008] The second technical solution of the present invention is a DNA molecule encoding the recombinant protein PME, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] The third technical solution of the present invention is a recombinant vector comprising the DNA molecule.

[0010] The fourth technical solution of the present invention is an engineered bacterium comprising the DNA molecule or the recombinant vector.

[0011] The fifth technical solution of the present invention is the use of the recombinant protein PME, the DNA molecule, the recombinant vector or the engineered bacteria in the preparation of a multi-epitope genetically engineered subunit vaccine against porcine Pasteurella multocida.

[0012] The sixth technical solution of the present invention is a multi-epitope genetically engineered subunit vaccine of porcine Pasteurella multocida, comprising the recombinant protein PME.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] The multi-epitope genetically engineered subunit vaccine for porcine Pasteurella multocida provided by the present invention has the advantages of good safety, low cost, ease of use, and easy differentiation between naturally infected animals and immunized animals, and has good application prospects. The recombinant tandem epitope antigens of the present invention are used to prepare the vaccine without the need to culture pathogenic microorganisms. The recombinant tandem epitope antigens are derived from important structural proteins of porcine Pasteurella multocida and can effectively protect porcine Pasteurella multocida. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0016] Figure 1 This is the SDS-PAGE electrophoresis result of the induced expression of the recombinant protein PME of the present invention.

[0017] Figure 2 This is a diagram showing the purification analysis results of the recombinant protein PME of the present invention.

[0018] Figure 3 This is a diagram showing the Western Blot identification results of the recombinant protein PME after purification of the present invention.

[0019] Figure 4 This is a statistical graph of the test results of the ELISA method for measuring the serum antibody level of immunized mice.

[0020] Figure 5 This is a graph showing the bacterial load detection results of the mouse lung tissue after the virus attack according to the present invention.

[0021] Figure 6 This is a graph showing the survival of mice after the poison challenge of the present invention.

[0022] Figure 7 This is a picture of the lung tissue lesions of mice after being challenged with poison according to the present invention. DETAILED DESCRIPTION

[0023] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] 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 without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0029] An embodiment of the present invention provides a recombinant protein PME, whose amino acid sequence is shown in SEQ ID NO.1.

[0030] The embodiment of the present invention also provides a DNA molecule encoding the recombinant protein PME, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0031] An embodiment of the present invention also provides a recombinant vector comprising the DNA molecule.

[0032] In some specific embodiments, the recombinant vector is pET30a-PME.

[0033] The embodiment of the present invention further provides an engineered bacterium comprising the DNA molecule or the recombinant vector.

[0034] In some specific embodiments, the engineered bacteria is BL21-pET30a-PME.

[0035] The embodiments of the present invention also provide the use of the recombinant protein PME, the DNA molecule, the recombinant vector or the engineered bacteria in preparing a recombinant tandem epitope antigen vaccine against porcine Pasteurella multocida.

[0036] The embodiment of the present invention further provides a porcine Pasteurella multocida recombinant tandem epitope antigen vaccine, comprising the recombinant protein PME.

[0037] In some specific embodiments, a clinically acceptable adjuvant is also included.

[0038] In some specific embodiments, the adjuvant comprises Montanide GEL 01PR.

[0039] The present invention creatively screens B cell antigen epitopes and T cell antigen epitopes that can bind to MHC-I and MHC-II molecules through bioinformatics methods, and then further integrates the antigen epitopes into recombinant proteins, explores the feasibility of developing a Pm multi-epitope vaccine, and evaluates the immune effect of the multi-epitope vaccine through mouse immunization, providing new ideas for the development of a new porcine Pasteurella multocida vaccine.

[0040] The present invention selected six key structural proteins of Pm through literature research. Bioinformatics analysis software was used to analyze and screen the amino acid sequences encoded by these key structural protein genes. T cell antigen epitopes capable of binding to MHC class I / II molecules and B cell antigen epitopes of peptide or protein surface residues capable of binding to antibodies were screened from the selected virulence factors. Multi-epitope fusion antigen genes were constructed and synthesized and expressed in Escherichia coli. The antigen epitopes were integrated into a recombinant protein, PME, and mixed with an adjuvant to produce a multi-epitope genetically engineered subunit vaccine for porcine Pasteurella multocida. This vaccine offers advantages such as good safety, low cost, ease of use, and easy differentiation between infected and immunized animals, thus promising promising applications.

[0041] The specific steps are as follows:

[0042] (1): The suitable candidate antigen genes in the genome of Pasteurella multocida were screened through literature search: PlpE, OmpA, OmpH, VacJ, Omp87, and Cp39;

[0043] (2): Using bioinformatics software, the amino acid sequences encoded by the genes of important structural proteins of Pasteurella multocida were analyzed and screened to identify T cell antigen epitopes that can bind to MHC class I and MHC class II molecules;

[0044] (3): Using bioinformatics software to analyze the amino acid sequences encoded by the important structural protein genes of porcine Pasteurella multocida to predict B cell antigen epitopes;

[0045] (4): The antigen epitope sequences obtained by screening are connected in series through a flexible linker to obtain the optimal epitope combination to obtain the recombinant protein PME. The constructed recombinant plasmid is transformed into BL21 competent cells, and the PME protein is obtained through culture, extraction and purification.

[0046] (5): The recombinant protein PME is mixed with an adjuvant at a certain concentration to obtain a multi-epitope genetically engineered subunit vaccine against porcine Pasteurella multocida.

[0047] The antigen epitopes that can bind to MHC-Ⅱ molecules are 16 short peptides, namely DVNRVGSEY, FIYSVLSDV, YIYAIKPDA, GIYGEIAQL, DIGSVTAGL, FMPELALRV, GDDVGVSDY, AINFKSAEF, LLEQSQDPY, TMWDFNYKV, VMLPLYGPA, YLDRGYAQF, GSDQVDVIY, STTAFAAPF, GDDVGLSDY and FAYEGLGTL.

[0048] The antigen epitopes that can bind to MHC-I class molecules are 10 short peptides, namely DVNRVGSEY, AVELGYDDF, GDDVGVSDY, AINFKSAEF, LLEQSQDPY, YLDRGYAQF, GSDQVDVIY, QTDAWWKLF, STTAFAAPF and GDDVGLSDY.

[0049] The B cell antigen epitopes are 21 short peptides, namely SEPSSAP, SQQSSFK, QPSADYK, VRSDYKVYDKEPA, THSTQVSP, VDYRPDI, NKCDSVK, SYSPPLR, QSQDPYI, KVSTPKQ, HYNSVGRY, QAFSSSK, YPLDREH, YRTRQCPDSY, NVPDYSDP, YSDPSRVRA, KPLKKYQG, GLSDYTY, AGYSQKYVKQ, VEQNPPA and DYAQSKV.

[0050] Preferably, the step of further screening B cell antigen epitopes and T cell antigen epitopes that can bind to MHC class I / II from the screened virulence factors is:

[0051] The amino acid sequences of the above six candidate genes were downloaded from NCBI for B cell antigen epitope prediction;

[0052] T cell antigen epitopes that can bind to MHC-I / II molecules are directly predicted from virulence factors, and their immunogenicity is calculated. Positive short peptides with IC50 less than or equal to 500 are selected as antigen epitopes that bind to MHC-I / II molecules.

[0053] The antigen epitope sequence obtained by screening was fused and expressed through the linker "GSG" to form a complete protein sequence. The complete PME protein sequence is shown in SEQ ID NO.1.

[0054] After optimization and synthesis by Bioengineering, a subunit vaccine based on prokaryotic expression of Pm antigen epitope was developed. The PME recombinant antigen gene sequence is SEQ ID NO.2, and the recombinant antigen gene sequence is 1287bp.

[0055] The complete protein sequence was analyzed and tested for antigenicity, toxicity, allergic response, signal peptide, hydrophilicity, surface accessibility and flexibility, and the combination sequence with relatively strong antigenicity was selected as the optimal combination of tandem epitopes.

[0056] The sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. (hereinafter referred to as Sangon) for synthesis.

[0057] Restriction sites Nde I and Xho I were introduced into the two segments of the sequence, and protective bases were added to improve cleavage efficiency. The expression vector is pET-30a(+), which carries an N-terminal His tag / Thrombin restriction site / S tag / EK protease tag and an optional C-terminal His tag.

[0058] The constructed recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells, and the PME protein was obtained through culture, induced expression and purification.

[0059] Through gene cloning technology, the exogenous target gene is constructed into an expression vector and introduced into the expression strain, so that it is expressed in a specific prokaryotic organism as a subunit vaccine, which is used to stimulate the body to produce specific antibodies against porcine Pasteurella multocida.

[0060] Example 1

[0061] Preparation method and steps of porcine Pasteurella multocida multi-epitope genetically engineered subunit vaccine:

[0062] 1. The suitable candidate antigen genes PlpE, OmpA, OmpH, VacJ, Omp87, and Cp39 in the Pm genome were screened through literature search;

[0063] The amino acid sequences of six candidate genes were downloaded in fasta format from the NCBI (https: / / www.ncbi.nlm.nih.gov) database, including the amino acid sequences of PlpE, OmpA, OmpH, VacJ, Omp87, and Cp39.

[0064] B cell linear epitopes were predicted using the Immune Epitope Database (IEDB), funded by the National Institute of Allergy and Infectious Diseases. Protein beta-sheet regions were predicted using the Chou & Fasman Beta-Turn Prediction method; protein surface accessibility was predicted using the Emini Surface Accessibility Prediction method; protein antigenic determinants were predicted using the semi-empirical Kolaskar & Tongaonkar Antigenicity method; protein flexibility regions were predicted using the Karplus & Schulz Flexibility Prediction method; and hydrophilicity regions were predicted using the Parker Hydrophilicity Prediction method. Linear B cell epitopes were predicted using the Bepipred Linear Epitope Prediction 2.0 artificial intelligence method, trained using epitope data from antibody-antigen-protein structure annotation. These methods were used to predict antigenic epitopes for six candidate antigenic proteins from Pasteurella multocida.

[0065] The Immune Epitope Database (IEDB), funded by the National Institute of Allergy and Infectious Diseases (NIAID), was used to predict T cell linear epitopes that bind to MHC class I / II molecules. Mouse and porcine MHC alleles were predicted using the SMM with a Peptide:MHC Binding Energy Covariance matrix (SMMPMBEC). Positive short peptides common to both mouse and porcine with an IC50 of ≤500 were selected as T cell antigen epitopes that bind to MHC class I / II molecules.

[0066] These predicted epitopes were concatenated using a flexible linker (GSG). The concatenated sequences were predicted using AllerTOPv.2, VaxiJen v2.0, and DNAstar software, indicating that the concatenated sequences had strong hydrophilicity, flexibility, a high antigenic index, and good surface accessibility. Bioinformatics tools such as ExPaSy ProtParam, SignalP-6.0Server, DeepTMHMM Server, SOPMA Server, IEDB, and SYFPEGII were used to analyze the basic physicochemical properties, signal peptides, transmembrane domains, and secondary structures of the concatenated sequences to further determine their B cell and T cell epitopes. A combination sequence with relatively strong antigenicity was selected as the optimal combination of concatenated epitopes.

[0067] The above B cell antigen epitopes and T cell antigen epitopes that bind to MHC class I and MHC class II molecules were combined, along with antigen epitopes with repeating sequences. These antigen epitopes were then connected using a "GSG" linker to form a complete protein sequence. The protein sequence was codon-optimized in E. coli and then fully synthesized. The synthesized gene was ligated between the Nde I and Xho I restriction sites of the pET-30a vector, retaining the 6×His tag at the C-terminus of the vector. The identified recombinant vector was named pET30a-PME.

[0068] The gene sequence of the biosynthesized PME is shown in SEQ ID NO. 2. The recombinant plasmid was transformed into the E. coli BL21(DE3) host, plated onto LB plates containing kanamycin, and cultured overnight in a 37°C incubator. This engineered strain, designated BL21-pET30a-PME, was used to express the recombinant protein.

[0069] SEQ ID NO.1:DVNRVGSEYGSGSEPSSAPGSGFIYSVLSDVGSGSQQSSFKGSG YIYAIKPDAGSGQPSADYKGSGVRSDYKVYDKEPAGSGAVELGYDDFGSGTHSTQVSPGSGGIYGEIAQLGSGVDYRPDIGSGDIGSVTAGLGSGNKCDSVKGSGFMPELALRVGSGGDDVGVSDYGSGAGYSQKYVKQGSGAINFKSAEFGSGDYAQSKVGSGLLEQSQDPYIGSGSYSPPLRGSGTMWDFNYKVGSGKVSTPKQGSGVMLPLYGPAGSGKVSTPKQGSGYLDRGYAQFGSGHYNSVGRYGSGQAFSSSKGSGGSDQVDVIYGSGYPLDREHGSGQTDAWWKLFGSGNVPDYSDPGSGYSDPSRVRAGSGSTTAFAAPFGSGKPLKKYQGGSGGDDVGLSDYGSGGLSDYTYGSGVEQNPPAGSGFAYEGLGTL。

[0070]

[0071] 2. Exploration and identification of expression conditions of recombinant protein PME

[0072] Take the above-mentioned engineered bacteria BL21-pET30a-PME transformed into E. coli BL21 (DE3) competent state and culture it in 5mL LB liquid medium overnight, add Kana antibiotic stock solution to make the final concentration 50μg / mL, and culture it in a shaker at 37℃ overnight. After expansion culture the next day, add 2mL of overnight cultured engineered bacteria BL21-pET30a-PME seed solution to 200mL LB medium, add 200μL Kana antibiotic, and culture it in a shaker at 37℃ until OD 600 The pH value was about 0.6. IPTG was added to a final concentration of 1 mM and induced for 5 hours at 37°C. 1 mL of whole bacteria was collected before and after induction. After 5 hours of IPTG induction, the cells were collected by centrifugation at 8000 rpm at 4°C for 3 minutes. The supernatant was discarded and 10 mL of Binding Buffer was added to resuspend the cells. The centrifuge tube containing the resuspended liquid was placed on an ice-water mixture to maintain a low temperature and ultrasonically disrupted: ultrasonically disrupted for 3 seconds each time, with a 2-second interval, at 30% power, until the resuspended liquid was broken into clarity and translucency. 100 μL of the disrupted whole bacteria was collected and stored at -20°C. The ultrasonically disrupted bacterial solution was centrifuged at 11000 rpm at 4°C for 20 minutes and the supernatant was collected. The precipitate was resuspended in 500 μL of Binding Buffer.

[0073] Purification of PME protein: The precipitate collected by crushing and centrifugation was resuspended in inclusion body washing buffer, stirred and washed for 30 minutes, centrifuged at 12000 rpm at 4°C for 15 minutes, the supernatant was discarded, the precipitate was resuspended in PBS, centrifuged at 12000 rpm at 4°C for 10 minutes, and repeated three times to collect the precipitate.

[0074] SDS-PAGE detection of protein expression location: Take 20 μL of the whole bacteria before and after induction, the whole bacteria after induction disruption, the supernatant, the precipitate, and the purified precipitate, then add 5 μL of 5× Loading Buffer, vortex and spin, heat and boil at 100℃ for 10 minutes to fully denature the protein, and perform 12% SDS-PAGE. After staining with Coomassie Brilliant Blue, the results are as follows: Figure 1 、 2 shown.

[0075] Western blot analysis of PME protein: 20 μL of the purified pellet was added to 5 μL of 5× Loading Buffer. After vortexing briefly, the protein was fully denatured and then subjected to 12% SDS-PAGE. After electrophoresis, transfer to a membrane was performed: After cutting the PVDF membrane and gel to the appropriate size, the membrane was activated by soaking in methanol for 1 minute. The PVDF membrane was then transferred to deionized water and soaked for 1 minute, followed by equilibration in transfer buffer for 2 minutes. Filter paper and sponge were soaked in transfer buffer. The membrane was assembled in the order of positive electrode, sponge, three layers of filter paper, PVDF membrane, gel, three layers of filter paper, sponge, and negative electrode. The clamp was closed and placed in a wet transfer tank. No bubbles were allowed. Transfer was performed on ice at 100 V for 25 minutes. Blocking: The membrane was rinsed with TBST for 2 minutes to remove the transfer buffer. Blocking was performed by adding 5% skim milk and shaking for 2 hours. Incubate the primary antibody: rinse the transferred protein membrane with washing solution TBST for 2 minutes, dilute the anti-His antibody 10,000 times with 5% skim milk with the primary antibody diluent, and incubate at 4°C overnight. Incubate the secondary antibody: take out the transferred protein membrane and soak it in TBST solution to wash 3 times, 15 minutes each time, dilute the HRP-labeled goat anti-mouse secondary antibody 10,000 times with TBST containing 5% skim milk, and incubate at room temperature for 2 hours. Color development: take out the transferred protein membrane and soak it in TBST solution to wash 3 times, 15 minutes each time, mix equal amounts of developer A and B, and add them to the membrane for development. Western blot results are as follows: Figure 3 shown.

[0076] The purified protein was sequenced, and the sequencing result was consistent with SEQ ID NO.1.

[0077] The purified target protein was assayed for protein concentration using a BCA protein concentration assay kit and the protein concentration was adjusted to 2.5 mg / mL. The protein was then aliquoted and stored at -80°C as an immunogen for animal immunization experiments.

[0078] Example 2

[0079] 1. Animal Immunization Experiment Grouping and Immunogen Preparation

[0080] Sixty-four 6-week-old female BALB / c mice were selected, of which 32 were used to detect the survival of mice after challenge and divided into 4 groups of 8 mice each, as follows: (1) Pm D inactivated vaccine group; (2) PME immunization experimental group; (3) adjuvant group; (4) PBS group. The remaining 32 were used to detect the bacterial load in the lung tissue of mice after challenge and divided into 4 groups of 8 mice each, as follows: (1) Pm D inactivated vaccine group; (2) PME immunization experimental group; (3) adjuvant group; (4) PBS group.

[0081] The immunogen preparation method for each group is as follows:

[0082] (1) Pm D inactivated vaccine group: 2 mL of clinically isolated porcine Pasteurella multocida PM-D2405 (isolated from a pig farm in Jiangling County, Jingzhou City, Hubei Province) was inoculated into 200 mL of BHI liquid medium containing newborn calf serum and cultured at 37°C and 180 rpm until the OD 600 When the concentration is 0.6, 100 μL is diluted and plated for counting Pasteurella suis PM-D2405. Add 0.4 mL of formaldehyde to the remaining bacterial suspension and inactivate it at 37°C for 18 hours, shaking the culture 2-3 times. After inactivation, 200 μL of the suspension is plated on a BHI plate containing newborn calf serum to verify inactivation. The thoroughly inactivated Pasteurella suis PM-D2405 is emulsified with Montanide GEL01PR adjuvant at a ratio of 10:1 by volume, and 100 μL is injected into each mouse for each immunization.

[0083] (2) PME immunization experimental group: The recombinant protein PME storage solution was diluted to 2.5 mg / mL and emulsified with MontanideGEL 01PR adjuvant at a volume ratio of 10:1. Each mouse was immunized with 100 μL each time.

[0084] (3) Adjuvant group: Montanide GEL 01PR adjuvant was emulsified with sterile PBS buffer at a volume ratio of 1:10, and 100 μL was injected into each mouse for each immunization.

[0085] (4) PBS group: sterile PBS buffer, 100 μL per mouse for each immunization injection.

[0086] 2. Mouse immunization

[0087] Mice were immunized on days 0, 14, and 28, and multiple immunizations were performed subcutaneously on the back of each group of mice. For the PM-D2405 inactivated vaccine group, the colony counts for the three immunizations were 3.9×10 8 CFU, 3.6×10 8 CFU and 3.5 × 10 8 CFU.

[0088] On the 35th day, each mouse in each group was intraperitoneally injected with 100 μL of Pasteurella suis PM-D2405, with a viable bacterial count of 1.04×10 7 CFU.

[0089] 3. Animal Experiment Sample Collection

[0090] Serum was collected on the 0th day as a negative control. Blood was collected from the tail vein of each group of mice on the 14th, 28th, and 35th days. The blood was placed at room temperature for 1 hour and then at 4°C overnight. The next day, the blood was placed in a 4°C centrifuge at 2000g for 20 minutes, and the supernatant was aspirated. This process was repeated once and then stored at -20°C.

[0091] 4. Detection of serum antibody levels in mice after immunization

[0092] In order to evaluate the immune response of mice to the recombinant protein PME, the indirect ELISA method was used to detect the antibody level. The specific operation method is as follows:

[0093] (1) Coating: The overnight culture of Pasteurella suis PM-D2405 was centrifuged at 3000 g for 5 min to collect the cells. The cells were resuspended three times with antigen coating solution and then resuspended to 10 mL with antigen coating solution. The cells were disrupted with an ultrasonic disruptor until the cells became clear. The protein concentration was determined by BCA protein quantification. The bacterial disruption solution was diluted to 100 μg / mL and 100 μL per well was coated on a 96-well plate. The plates were coated at 4°C overnight.

[0094] (2) Blocking: The next day, the liquid in the plate was shaken off, patted dry, and washed three times with PBST, each time for 5 minutes. 150 μL of antigen coating solution containing 2% BSA was added to each well and incubated at 37°C for 2 hours.

[0095] (3) Incubation with primary antibody: Shake off the liquid in the plate, pat dry, and wash three times with PBST, 5 minutes each time. Dilute the serum collected at different time points 200 times with antibody diluent, and make a continuous 1:2 dilution on the ELISA plate, 100 μL per well, and perform three replicates for each serum. At the same time, set up a negative control, and incubate at 37°C for 1 hour.

[0096] (4) Incubation with secondary antibody: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 min each time. Add 100 μL of HRP secondary antibody to each well at a dilution of 1:5000 and incubate at 37°C for 1 h.

[0097] (5) Add substrate: Shake off the liquid in the plate, pat dry, and wash three times with PBST, 5 minutes each time. Add 100 μL of TMB substrate buffer to each well and incubate at room temperature for 5 minutes.

[0098] (6) Termination: Add 50 μL 1M H2SO4 to each well to terminate the reaction, and read the OD on a microplate reader within 15 minutes. 450 The antibody level changes, the results are as follows Figure 4 As shown, the antibody level in the PME protein group was much higher than that in the adjuvant group and PBS group, but slightly lower than that in the inactivated vaccine group.

[0099] 5. Mouse challenge

[0100] Virus challenge: Remove swine Pasteurella multocida PM-D2405 from -80℃, streak the plate and incubate at 37℃ for 24h, pick a single colony and transfer it to 5mL BHI liquid medium containing newborn calf serum, and incubate at 180rpm at 37℃ overnight. Inoculate the overnight culture solution into 10mL BHI liquid medium containing newborn calf serum at a dilution of 1:100, and incubate at 180rpm at 37℃ until OD 600 The OD value is about 0.6. Take 100 μL of bacterial solution and add it to 0.9% saline to dilute it and spread it on BHI plate containing newborn calf serum to count the colonies. Continue for three days to make the colony number stable. On the second day (35 days), culture the bacterial solution in large quantities and culture it to OD 600 The bacteria were diluted with sterilized 0.9% saline and challenged with mice. The colony count was performed at the same time. Each mouse in each group was injected intraperitoneally with 100 μL of porcine Pasteurella PM-D2405. The number of viable bacteria was 1.04×10 7 CFU. Record the time of infection and the time of death of each group of mice, and record the survival rate after 7 days of observation. The results are as follows Figure 5 Mice were killed 8 hours after infection for detection of bacterial load in lung tissue. Figure 6 shown.

[0101] Lung tissue was collected and fixed in 10% neutral buffered formalin. It was sent to a biological company for embedding, HE tissue sections were stained and examined by light microscopy. Lung tissue was assessed as mild (1 point), moderate (2 points), severe (3 points), extremely severe (4 points), and no lesions (0 points). The range of lung lesions included inflammatory cell infiltration, hemorrhage, and lung lesions (none, single lesions, multiple lesions, and localized extensive). The results were as follows: Figure 7 shown.

[0102] Depend on Figures 5-7 As shown, the survival rate of the PME protein group was much higher than that of the adjuvant and PBS groups, and was comparable to that of the inactivated vaccine group. The bacterial load in the lung tissue of mice in the PME protein group after challenge was much lower than that in the adjuvant and PBS groups, but higher than that in the inactivated vaccine group. Lung lesions in the PME protein group were milder, similar to those in the inactivated vaccine group, while lung tissue in the adjuvant and PBS groups showed predominantly inflammatory cell infiltration.

[0103] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A recombinant protein PME, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. The DNA molecule encoding the recombinant protein PME according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that Comprising the DNA molecule according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector is pET30a-PME.

5. An engineered bacterium, characterized in that: Comprising the DNA molecule according to claim 2 or the recombinant vector according to claim 3.

6. The engineered bacteria according to claim 5, characterized in that The engineered bacteria is BL21-pET30a-PME.

7. Use of the recombinant protein PME according to claim 1, the DNA molecule according to claim 2, the recombinant vector according to claim 3, or the engineered bacteria according to claim 5 in the preparation of a multi-epitope genetically engineered subunit vaccine against porcine Pasteurella multocida.

8. A porcine Pasteurella multocida multi-epitope genetically engineered subunit vaccine, characterized in that: Comprising the recombinant protein PME according to claim 1.

9. The porcine Pasteurella multocida multi-epitope genetically engineered subunit vaccine according to claim 8, characterized in that: Clinically acceptable adjuvants are also included.

10. The porcine Pasteurella multocida multi-epitope genetically engineered subunit vaccine according to claim 9, characterized in that: The adjuvants included Montanide GEL 01PR.

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