Preparation method and application of pig pasteurella multocida multi-epitope genetic engineering subunit vaccine

Abstract

The invention discloses a preparation method and application of a pig pasteurella multocida multi-epitope genetic engineering subunit vaccine, and belongs to the field of genetic engineering in biotechnology. Comprising the following steps: searching and selecting six Pm important structural proteins through literature, analyzing and screening sequences of gene coding amino acids of the Pm important structural proteins through bioinformatics analysis software, and screening T cell epitopes capable of being combined with MHC-I / II molecules from screened virulence factors, and a B cell antigen epitope of a peptide or protein surface residue capable of being combined with an antibody, constructing and synthesizing a multi-epitope fusion antigen gene, and expressing the multi-epitope fusion antigen gene in escherichia coli. The swine pasteurella multocida multi-epitope genetic engineering subunit vaccine is obtained by integrating antigen epitopes into recombinant protein PME and mixing the recombinant protein PME with an adjuvant, has the advantages of good safety, low cost, simplicity and convenience in use and easiness in distinguishing infected animals from immune animals, and has a good application prospect.

Description

Technical Field

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

[0002] Swine pasteurellosis is an acute epidemic or sporadic and secondary infectious disease caused by Pasteurella multocida (Pm), with fibrinous pleuropneumonia, pharyngitis and sepsis as the main characteristics. Sick pigs will have obvious high fever, asthma, dyspnea, loss of appetite, etc. The most acute infection often presents clinical symptoms of sepsis, causing the sick pigs to die within 1 day. The disease is highly contagious, harmful, has a fast transmission speed and a high mortality rate, which seriously affects the growth and development of pigs in large-scale pig farms and hinders the development of pig farms and pig farming. The disease is often mixed with other diseases, such as swine erysipelas and swine streptococcosis, making the condition more complicated and more difficult to cure sick pigs. With the abuse of antibiotics, Pm-resistant strains have appeared in large numbers, and the spectrum of resistance is becoming wider and wider, making conventional antibiotics unable to treat, which makes immune prevention an important way to prevent and control the disease.

[0003] Inactivated vaccines are prepared by inactivating bacteria through exogenous methods such as heating, drying, and formaldehyde, and mixing with adjuvants to prepare corresponding vaccines. They are one of the effective ways to prevent Pm. Generally, the inactivated vaccine strain is selected first, the local dominant epidemic strain is isolated and cultured, and then treated by physical and chemical methods to make it lose the ability to cause pathogenic infection but still maintain good immunogenicity. Finally, the immune adjuvant is selected for emulsification and preparation, which can effectively reduce the mortality rate of pigs. Inactivated vaccines have poor cross protection, long immune response, short antibody maintenance time, and require multiple immunizations. Attenuated live vaccines are Pm strong strains that are artificially destroyed by chemical and physical methods. The antigenic components such as capsular polysaccharides are lost or mutated, and finally the attenuated strains are screened. The attenuated vaccine is in danger of relapse and has great hidden dangers. Therefore, it is urgent to develop a new vaccine that can effectively prevent pasteurellosis.

[0004] Gene engineered subunit vaccine is a vaccine made by purifying protein antigens expressed by genetic engineering. Subunit vaccine is a vaccine that uses genetic engineering technology to connect the antigen gene of porcine 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 of 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 the advantages of good safety, low cost, ease of use, and easy distinction between infected animals and immune animals, and have good application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a multi-epitope genetic engineering 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, and its nucleotide sequence 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] A fourth technical solution of the present invention is an engineered bacterium comprising the DNA molecule or the recombinant vector.

[0011] A 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, easy use and easy distinction 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 vaccines, and there is no need to cultivate pathogenic microorganisms. The recombinant tandem epitope antigens are derived from important structural proteins of porcine Pasteurella multocida and can play an effective protective role against 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 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.

[0016] Figure 1 The figure 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 results of Western Blot identification 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 of the present invention for measuring the serum antibody level of immune mice.

[0020] Figure 5 This is a graph showing the results of detecting the bacterial load in the lung tissue of mice after challenge with the virus according to the present invention.

[0021] Figure 6 This is a graph showing the survival of mice after the virus attack 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments 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. Each smaller range between the intermediate value in any stated value or stated range and 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.

[0025] 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 materials related to the documents. In the event of a conflict with any incorporated document, the content 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 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 application description and examples are exemplary only.

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

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

[0029] The 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, and its nucleotide sequence is shown as SEQ ID NO.2.

[0031] The 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 also 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 the preparation of a recombinant tandem epitope antigen vaccine against porcine Pasteurella multocida.

[0036] The embodiment of the present invention also 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 out 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 selects 6 important structural proteins of Pm through literature search, analyzes and screens the sequences of amino acids encoded by the genes of the important structural proteins of Pm through bioinformatics analysis software, screens 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, constructs and synthesizes multi-epitope fusion antigen genes and expresses them in Escherichia coli. The antigen epitopes are integrated into recombinant protein PME and mixed with adjuvants to obtain a multi-epitope genetic engineering subunit vaccine for porcine Pasteurella multocida, which has the advantages of good safety, low cost, simple use, and easy distinction between infected animals and immune animals, and has good application prospects.

[0041] The specific steps are as follows:

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

[0043] (2): The amino acid sequences encoded by the important structural protein genes of porcine Pasteurella multocida were analyzed and screened by bioinformatics software to identify T cell antigen epitopes that can bind to MHC-I and MHC-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, and 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] There are 16 short peptides that can bind to MHC-Ⅱ molecules, 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-I / II class 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 prepared by fusion expression 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 combined sequence with relatively strong antigenicity was selected as the best combination of tandem epitopes.

[0056] The sequence was sent to Shanghai Biotech Co., Ltd. (hereinafter referred to as Shanghai Biotech) 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 the cleavage efficiency. The expression vector is: pET-30a (+) with 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 expressed in a specific prokaryotic organism by constructing an expression vector and introducing it into an expression strain. The subunit vaccine is used to stimulate the body to produce specific antibodies against porcine Pasteurella multocida.

[0060] Example 1

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

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

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

[0064] The Immune Epitope Database (IEDB) funded by the National Institute of Allergy and Infectious Diseases of the United States was used to predict B cell linear epitopes. The Chou & Fasman Beta-Turn Prediction method was used to predict the β-folding region of the protein; the Emini Surface Accessibility Prediction method was used to predict the surface accessibility of the protein; the semi-empirical method Kolaskar & Tongaonkar Antigenicity was used to predict the antigenic determinant of the protein; the Karplus & Schulz Flexibility Prediction method was used to predict the protein flexibility region; the Parker Hydrophilicity Prediction method was used to predict the hydrophilic region; the Bepipred Linear Epitope Prediction 2.0 artificial intelligence method trained with epitope data annotated by antibody-antigen-protein structure was used to predict linear B cell epitopes. The above methods were used to predict the antigenic epitopes of 6 candidate antigenic proteins of Pasteurella multocida.

[0065] The Immune Epitope Database (IEDB) funded by the National Institute of Allergy and Infectious Diseases of the United States was used to predict T cell linear epitopes that can bind to MHC-I / II molecules. The SMM with a Peptide:MHC Binding Energy Covariance matrix (SMMPMBEC) was used to predict mouse and pig MHC alleles, and the positive short peptides common to mice and pigs were selected as T cell antigen epitopes that bind to MHC-I / II molecules when the IC50 was less than or equal to 500.

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

[0067] Combine the above B cell antigen epitopes and T cell antigen epitopes that can bind to MHC-I and MHC-II molecules and combine the antigen epitopes with repeating sequences, and then connect these antigen epitopes with "GSG" linkers to form a complete protein sequence. After the protein sequence is optimized for E. coli codons, the whole gene is synthesized, and the synthesized gene is connected to the restriction sites of Nde I and Xho I of the pET-30a vector, retaining the 6×His tag at the C-terminus of the vector, and the identified correct recombinant vector is named pET30a-PME.

[0068] The gene sequence of the PME synthesized by Bioengineering is shown in SEQ ID NO. 2. The recombinant plasmid was transformed into the E. coli BL21 (DE3) host, then spread on an LB plate containing kanamycin and cultured overnight in a 37°C bacterial constant temperature incubator. It was used as an engineering bacterium expressing recombinant protein and named BL21-pET30a-PME.

[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 37℃ shaker overnight. After expansion culture the next day, add 2mL of the overnight cultured engineered bacteria BL21-pET30a-PME seed solution to 200mL LB medium, add 200μL kana antibiotics, and culture it in a 37℃ shaker until OD 600 The pH value is about 0.6. Add IPTG with a final concentration of 1mM and induce expression at 37℃ for 5h. Collect 1mL of whole bacteria before and after induction respectively. After IPTG induction for 5h, centrifuge at 4℃, 8000rpm, 3min to collect the bacteria, discard the supernatant, and add 10mL Binding Buffer to resuspend the bacteria; place the centrifuge tube containing the resuspended liquid on an ice-water mixture, keep it at a low temperature, and perform ultrasonic disruption: ultrasonic for 3s each time, 2s interval, and 30% power until the resuspended liquid is broken to clear and transparent. Collect 100μL of the broken whole bacteria and store at -20℃. Centrifuge the bacterial solution after ultrasonic disruption at 11000rpm, 4℃ for 20min, and collect the supernatant. Resuspend the precipitate with 500μL 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 4°C 12000 rpm for 15 minutes, the supernatant was discarded, the precipitate was resuspended in PBS, centrifuged at 4°C 12000 rpm 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, the supernatant, the precipitate, and the purified precipitate, and then add 5 μL of 5× Loading Buffer. After vortexing, heat and boil at 100℃ for 10 minutes to fully denature the protein and perform 12% SDS-PAGE. After Coomassie Brilliant Blue staining, the results are as follows: Figure 1 , 2 shown.

[0075] Western blot identification of PME protein: Take 20 μL of the purified precipitate, add 5 μL 5×LoadingBuffer, vortex and separate, heat and boil at 100℃ for 10min, and perform 12% SDS-PAGE after fully denaturing the protein. After the electrophoresis, transfer to the membrane: Cut the PVDF membrane and gel to the appropriate size, and activate it by soaking the PVDF membrane in methanol for 1min. Then transfer the PVDF membrane to deionized water and soak it for 1min and then transfer it to the transfer buffer for 2min. Soak the filter paper and sponge in the transfer buffer, assemble them in the order of positive electrode, sponge, 3 layers of filter paper, PVDF membrane, gel, 3 layers of filter paper, sponge, and negative electrode, close the clamp and put it in the wet transfer tank. There should be no bubbles, and transfer the membrane on ice at 100V for 25min. Blocking: Rinse the protein membrane with washing solution TBST for 2min to wash off the transfer solution on the membrane. Add 5% skim milk for blocking, and block on a shaker for 2h. 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 measured for protein concentration using a BCA protein concentration assay kit and the protein concentration was adjusted to 2.5 mg / mL. Finally, it was 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] 64 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, 8 mice in each group, 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, 8 mice in each group, 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 PM-D2405 (isolated from a pig farm in Jiangling County, Jingzhou City, Hubei Province) was inoculated into 200 mL of BHI liquid culture medium containing newborn calf serum and cultured at 37°C and 180 rpm until OD 600 The concentration of 100 μL of the culture medium was 0.6, and 100 μL was taken for dilution and plate application to calculate the number of porcine Pasteurella PM-D2405. 0.4 mL of formaldehyde was added to the remaining bacterial solution, and it was inactivated at 37°C for 18 hours, during which the culture medium was shaken 2-3 times. After the inactivation, 200 μL of the bacterial solution was spread on a BHI plate containing newborn calf serum to test the inactivation effect. After the completely inactivated porcine Pasteurella PM-D2405 was emulsified with Montanide GEL01PR adjuvant at a volume ratio of 10:1, 100 μL was injected into each mouse for immunization each time.

[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 and sterile PBS buffer were emulsified 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. PM-D2405 inactivated vaccine group: The colony counts of the three immunizations were 3.9×10 8 CFU, 3.6×10 8 CFU and 3.5 × 10 8 CFU.

[0088] Virus challenge: On the 35th day, each mouse in each group was intraperitoneally injected with 100 μL of swine Pasteurella PM-D2405, with a live 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 effect of mice on 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 cultured bacterial suspension of swine Pasteurella multocida PM-D2405 was centrifuged at 3000 g for 5 min to collect the bacteria, which were resuspended three times with antigen coating solution and then resuspended to 10 mL with antigen coating solution. The bacterial suspension was disrupted using an ultrasonic disruptor until the bacterial suspension became clear, and the protein concentration was determined by the BCA protein quantification method. 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 overnight at 4°C.

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

[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 1:2 dilution on the ELISA plate continuously, 100 μL per well, and perform 3 replicates for each serum. Set up a negative control at the same time, 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. Dilute HRP secondary antibody at 1:5000 and add 100 μL per well to the plate and incubate at 37°C for 1 h.

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

[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 min. 450 Value. Antibody level changes, results such as Figure 4 As shown, the antibody level in the PME protein group was much higher than that in the adjuvant group and the PBS group, but slightly lower than that in the inactivated vaccine group.

[0099] 5. Mouse challenge

[0100] Virus attack: Take out the porcine Pasteurella PM-D2405 from -80℃, streak the plate and incubate at 37℃ for 24h, pick a single colony and put it into 5mL BHI liquid medium containing newborn calf serum, and incubate it at 180rpm at 37℃ overnight. Inoculate the overnight cultured bacterial solution into 10mL BHI liquid medium containing newborn calf serum at a ratio of 1:100, and incubate it at 180rpm at 37℃ until OD 600 The OD value was about 0.6. 100 μL of bacterial solution was added to 0.9% saline solution and diluted on a BHI plate containing newborn calf serum for colony counting. This was continued for three days until the colony count became stable. On the second day (35 days), the bacterial solution was cultured in large quantities until the OD value reached 0. 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 intraperitoneally injected with 100 μL of porcine Pasteurella PM-D2405, and the number of live 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 the challenge to detect the bacterial load in lung tissue. The results are shown in Figure 6 shown.

[0101] Lung tissues were collected and fixed in 10% neutral buffered formalin. They were sent to a biological company for embedding, HE tissue sections were stained and examined by light microscopy. Lung tissues were evaluated 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 to 7 It can be seen that the survival rate of the PME protein group was much higher than that of the adjuvant group and the PBS group, and was not much different from 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 group and the PBS group, but higher than that in the inactivated vaccine group. The lung tissue lesions in the PME protein group were milder, similar to those in the inactivated vaccine group, while the lung tissues in the adjuvant group and the PBS group mainly showed inflammatory cell infiltration.

[0103] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope 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. A 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 multi-epitope genetically engineered subunit vaccine for porcine Pasteurella multocida, 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 multi-epitope genetically engineered subunit vaccine of porcine Pasteurella multocida according to claim 9, characterized in that: The adjuvants included Montanide GEL 01PR.

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