A recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae and its preparation and application

Abstract

The invention discloses a recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae and its preparation and application, and belongs to the field of genetic engineering. The amino acid sequence of the recombinant tandem epitope antigen of the invention is shown in SEQ ID NO.1, and its immune protection effect is close to that of porcine Actinobacillus pleuropneumoniae inactivated vaccine, and can be used to prepare porcine Actinobacillus pleuropneumoniae genetic engineering subunit vaccine, which has the advantages of good safety, low cost, simple use, easy distinction between infected animals and immune animals, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering in biotechnology, and particularly relates to a recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae and a preparation method thereof and application thereof in preparing a porcine Actinobacillus pleuropneumoniae vaccine. Background Art

[0002] Porcine contagious pleuropneumonia (PCP) is a contact respiratory infectious disease caused by Actinobacillus pleuropneumoniae (APP), characterized by fibrinous hemorrhagic and necrotizing pleuropneumonia, which can cause infection in pigs of all ages. The disease is often mixed with or secondary to other diseases, such as swine fever, blue ear disease, and streptococcal disease, making the condition more complicated and more difficult to cure sick pigs. APP inactivated vaccines can only target a single serotype, and even polyvalent vaccines can only cover a small number of serotypes; attenuated vaccines have the risk of reversion to toxicity, which poses a great hidden danger. On the other hand, with the abuse of antibiotics, APP-resistant strains have emerged in large numbers, and the spectrum of resistance is becoming wider and wider, making conventional antibiotics incapable of treatment, which also makes immune prevention an important way to prevent and control the disease.

[0003] 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 express the antigen gene of porcine Actinobacillus pleuropneumoniae on a vector. Peptide vaccine is also based on genetic engineering, selecting one or more antigen epitopes on porcine Actinobacillus pleuropneumoniae for expression in a recombinant vector. Antigens expressed by genetic engineering have high yield, high purity and good immunogenicity. The expression systems for expressing exogenous antigens mainly include bacteria, yeast, mammalian cells and insect cells. Genetically engineered subunit vaccines have good safety, and adjuvants are usually used to increase immunogenicity. 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

[0004] The purpose of the present invention is to provide a recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae and a preparation method thereof and application thereof in preparing a porcine Actinobacillus pleuropneumoniae vaccine.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] A gene encoding the recombinant tandem epitope antigen of Actinobacillus pleuropneumoniae, the nucleotide sequence of which is preferably as shown in SEQ ID NO.2.

[0008] A recombinant vector for expressing the recombinant tandem epitope antigen of Actinobacillus pleuropneumoniae, comprising the above coding gene. Furthermore, the recombinant vector uses pET-30a as a skeleton.

[0009] An engineered cell expressing the recombinant tandem epitope antigen of Actinobacillus pleuropneumoniae contains the above encoding gene or the above recombinant vector. Further, the engineered cell uses Escherichia coli BL21 Star (DE3) as a host cell.

[0010] The method for preparing the recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae comprises the following steps: culturing the above-mentioned engineered cells, inducing expression, and separating and purifying the recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae from the culture fluid.

[0011] The invention discloses an application of the recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae in preparing porcine Actinobacillus pleuropneumoniae vaccine.

[0012] A porcine Actinobacillus pleuropneumoniae vaccine comprises the porcine Actinobacillus pleuropneumoniae recombinant tandem epitope antigens and further comprises an adjuvant.

[0013] The porcine Actinobacillus pleuropneumoniae vaccine is a multi-epitope genetic engineering subunit vaccine.

[0014] Advantages and beneficial effects of the present invention: The recombinant tandem epitope antigens of porcine Actinobacillus pleuropneumoniae of the present invention are prepared into a multi-epitope genetic engineering subunit vaccine, which has the advantages of good safety, low cost, simple use, and easy distinction between naturally infected animals and immune 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 antigenic proteins expressed by pathogens in different periods and environments, which can improve the universal applicability of the vaccine and can also play an effective protective role against different serotype strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the electrophoresis result of double enzyme digestion of the recombinant expression vector pET-30a-AEP.

[0016] Figure 2 This is the SDS-PAGE electrophoresis result of the engineered bacteria BL21 Star-pET-30a-AEP induced expression.

[0017] Figure 3This is the Western Blot identification result of the recombinant protein AEP.

[0018] Figure 4 This is a diagram of the purification results of the recombinant protein AEP.

[0019] Figure 5 It is a graph showing the survival results of mice in each group after infection.

[0020] Figure 6 This is a statistical chart of the test results of ELISA method for determining the serum antibody level of immunized mice.

[0021] Figure 7 This is a picture of the lung tissue lesions of mice after infection. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

[0023] The present invention screens out B cell antigen epitopes and T cell antigen epitopes that can bind to MHC-I and MHC-II molecules by a bioinformatics method, selects recombinant tandem epitope antigens with the best antigenicity by combining these antigen epitopes, and evaluates the immune effect of the recombinant tandem epitope antigens by immunizing mice, and finds that the immune protection effect is close to that of the inactivated vaccine of porcine Actinobacillus pleuropneumoniae. The present invention provides new materials for the development of a new porcine Actinobacillus pleuropneumoniae vaccine.

[0024] Example 1 Design and preparation of recombinant tandem epitope antigens of Actinobacillus pleuropneumoniae

[0025] 1. Design of recombinant tandem epitope antigens of Actinobacillus pleuropneumoniae

[0026] Based on the results of previous studies, we selected the antigen genes ApxIA, ApxIIA, ApxIIIA, ApxIVA, TbpB, OmlA, and ApfA of APP to screen B cell antigen epitopes and T cell antigen epitopes that can bind to MHC-I and MHC-II molecules.

[0027] The amino acid sequences of ApxIA, ApxIIA, ApxIIIA, ApxIVA, TbpB, OmlA, and ApfA were downloaded using the NCBI (https: / / www.ncbi.nlm.nih.go) database.

[0028] 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; and the artificial intelligence method Bepipred Linear Epitope Prediction 2.0 trained based on epitope data annotated by antibody-antigen-protein structure was used to predict linear B cell epitopes. The above methods were used to predict antigenic epitopes for the above 7 antigenic proteins of Actinobacillus pleuropneumoniae.

[0029] The IEDB and NetMHC pred websites were used to predict T cell linear epitopes that can bind to MHC-I / II class molecules. SMM with a Peptide:MHC Binding Energy Covariance matrix (SMMPMBEC) was used to predict pig MHC alleles, and positive short peptides common to mice and pigs were selected as T cell antigen epitopes that bind to MHC-I / II class molecules when the ic50 was less than or equal to 500.

[0030] These predicted epitopes were concatenated through a flexible linker (GSG), and the sequences were predicted to have strong hydrophilicity, flexibility, high antigenic index and good surface accessibility using AllerTOPv.2, VaxiJen v2.0 tools and DNAstar software. The concatenated sequences were analyzed and tested for antigenicity, toxicity, allergic reaction, signal peptide, transmembrane structure, hydrophilicity, surface accessibility and flexibility using bioinformatics tools such as ExPaSy ProtParam, SignalP-6.0Server, DeepTMHMM Server, SOPMAServer, IEDB and SYFPEGII, and the combination sequences with relatively strong antigenicity were screened out.

[0031] The amino acid sequence of the recombinant tandem epitope antigen obtained through the above prediction and analysis screening is shown in SEQ ID NO.1, and it is named AEP protein.

[0032] 2. Preparation of recombinant tandem epitope antigens of Actinobacillus pleuropneumoniae

[0033] The AEP protein sequence was codon optimized for E. coli and then fully synthesized. The optimized nucleotide sequence is shown in SEQ ID NO. 2. Restriction sites Nde I and Xho I were introduced into the two segments of the synthesized sequence, and protective bases were added to improve the cutting efficiency. The sequence was cloned into the pET-30a vector by restriction digestion and ligation to obtain the recombinant expression vector pET-30a-AEP.

[0034] The recombinant expression vector pET-30a-AEP was double-digested with Nde I and Xho I, and the reaction system was shown in Table 1 below.

[0035] Table 1 Double enzyme digestion system

[0036]

[0037] After thorough mixing, the above reaction system was placed in a 37°C water bath for 2 hours of enzyme digestion reaction. After the reaction was completed, 1% agarose gel electrophoresis was performed and the results were observed. Figure 1 As shown, pET-30a-AEP was successfully constructed.

[0038] The recombinant expression vector pET-30a-AEP was transformed into the E. coli BL21 Star (DE3) host, and then coated with kanamycin LB plates and cultured overnight in a 37°C bacterial constant temperature incubator. It was used as an engineering bacterium expressing the recombinant protein AEP and named BL21 Star-pET-30a-AEP.

[0039] The engineered bacteria BL21 Star-pET-30a-AEP expressing the recombinant protein AEP was added to 5 mL of LB liquid medium containing 50 μg / mL of kanamycin and cultured overnight in a shaker at 37°C. After expansion the next day, 2 mL of the overnight cultured engineered bacteria seed liquid was added to 200 mL of TB medium containing 50 μg / mL of kanamycin and cultured in a shaker at 37°C until OD 600 About 0.8, add IPTG with a final concentration of 1mM to induce culture for 2h. Take 1mL of bacterial solution before and after IPTG induction, centrifuge at 4℃8000rpm for 3min to collect bacteria, discard the supernatant, 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, 30% power, until the resuspended liquid is broken to clarity and transparency. Centrifuge the bacterial solution after ultrasonic disruption at 4℃11000rpm for 30min, and collect the supernatant. Resuspend the precipitate with 500μL Binding Buffer.

[0040] SDS-PAGE detection of protein expression location: Take 20 μL of the whole bacteria before and after induction, the supernatant after induction, and the precipitate after induction saved in the above steps, then add 5 μL of 5× Loading Buffer, vortex for a moment, heat and boil at 100℃ for 10 minutes to fully denature the protein, and then perform 8% SDS-PAGE. The Coomassie Brilliant Blue staining results are as follows Figure 2 As shown, a protein with the same size as the target protein was successfully expressed.

[0041] Western blot identification of AEP protein: Collect the induced supernatant obtained in the previous step, heat and boil at 100℃ for 10min to fully denature the protein, and then perform 8% SDS-PAGE. After the electrophoresis, transfer the membrane: After cutting the PVDF membrane and gel to the appropriate size, soak the PVDF membrane in methanol for 1min to activate it. Then transfer the PVDF membrane to deionized water for 1min and then transfer it to the transfer buffer for 2min. Soak the filter paper and sponge in the transfer buffer, and transfer 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. Transfer the membrane on ice at 90V for 80min. Block: Rinse the protein membrane with washing solution TBST for 1-2min to wash off the transfer solution on the membrane. Add 5% skim milk for blocking and block on a shaker for 3h. Incubate the primary antibody: rinse the transferred protein membrane with washing solution TBST for 1-2 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, add them to the membrane, and develop them using a developer. Western blot results are as follows: Figure 3 As shown, it was proved that the prokaryotic expression of AEP protein was successfully achieved.

[0042] The above-mentioned induced supernatant was collected and purified AEP protein was obtained by nickel column affinity chromatography ( Figure 4 ). The purified target protein was ultrafiltered and then quantified using a BCA protein concentration assay kit and the protein concentration was adjusted to 2 mg / mL. Finally, it was packaged and stored at -80°C as an immunogen for animal immunization experiments.

[0043] Example 2 Animal Immunization Experiment

[0044] 1. Animal immunization experiment grouping and immunogen preparation

[0045] Thirty-two 6-week-old female BALB / c mice were randomly divided into the following four groups: (1) APP inactivated vaccine group; (2) AEP immunization experiment group; (3) adjuvant group; (4) PBS group. The immunogen preparation method and immunization conditions of each group are as follows:

[0046] (1) Inoculate 1 mL of APP type 1 CVCC259 strain into 100 mL of TSB liquid medium and culture at 37°C and 180 rpm until OD 600 The concentration of APP in the culture medium is about 0.6. Take 100 μL to dilute and plate. Add 0.2 mL of formaldehyde to the remaining bacterial solution and inactivate it at 37°C for 15-18 hours. During this period, shake the culture medium 2-3 times. After the inactivation is completed, take 200 μL to coat the TSB plate to test the inactivation effect. Emulsify the completely inactivated APP bacterial solution with an equal volume of Freund's adjuvant to obtain the inactivated APP vaccine. Each mouse is injected with 100 μL each time for immunization.

[0047] (2) The recombinant protein AEP stock solution was diluted to 2 mg / mL and emulsified with an equal volume of Freund's adjuvant to obtain the AEP vaccine. Each mouse was injected with 100 μL for each immunization.

[0048] (3) Freund's adjuvant and sterile PBS buffer were emulsified in equal volumes to obtain adjuvant control. Each mouse was injected with 100 μL each time for immunization.

[0049] (4) Sterile PBS buffer: 100 μL per mouse for each immunization injection.

[0050] The method of emulsification of recombinant protein solution, PBS, inactivated APP and Freund's adjuvant: add 2 mL of immunogen to a 10 mL syringe connected to a three-way tube, and then take 2 mL of Freund's adjuvant (v / v 1:1) to mix, connect two syringes to the two ends of the three-way tube, push the syringe back and forth until the liquid in the syringe stops flowing rapidly, and suck 10 μL of liquid and drop it on the cold water surface. If the droplets do not spread, it means that the emulsification is complete, and if the droplets spread, the emulsification continues. Store the emulsified sample on ice at 4°C.

[0051] 2. Mouse immunization

[0052] Mice were immunized on days 0, 14, and 28. The first immunization used Freund's complete adjuvant (primary immunization), and the next two immunizations used Freund's incomplete adjuvant (boosting immunization). Multiple immunizations were performed on each group of mice at the back subcutaneously. APP type 1 CVCC259 inactivated vaccine group: colony counts after three immunizations were 1.49×10 9 CFU, 1.53×10 9 CFU and 1.35 × 10 9 CFU.

[0053] Virus attack: The virus attack was carried out one week after the mice were boosted with immunization. The details are as follows: the APP 1 type CVCC259 strain was taken out from -80℃, streaked on the plate and cultured at 37℃ for 24h, then a single clone was picked and continued to be streaked on the plate, and a single clone was picked and cultured in 5mL of liquid TSB medium containing 10% primary bovine serum and 10μg / mL NAD at 180rpm at 37℃ overnight. Then the APP1 type CVCC259 strain was inoculated at 1:100 into 5mL of liquid TSB medium containing 10% primary bovine serum and 10μg / mL NAD at 180rpm at 37℃ and cultured at OD 600 About 0.6, take 100 μL of bacterial solution and sterilize with 0.9% NaCl 2 Dilute 10 -6 Spread TSB plates for colony counting for three consecutive days until the colony count stabilizes. On the second day (35 days), culture the bacterial solution in large quantities until the OD 600 About 0.6, sterilized 0.9% NaCl 2 The bacteria were diluted and challenged to mice, and the colony counts were performed. On the 35th day, each mouse in each group was intraperitoneally injected with 100 μL of APP type 1 CVCC259 live bacteria, containing 6.45×10 6 CFU. Record the time of infection and the time of death of each group of mice, and record the survival rate after 2 days of observation. The survival curve results are shown in Figure 5 As shown, the survival rate of the AEP 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.

[0054] Animal experiment sample collection: serum was collected on the 0th day as a negative control, and 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 2 hours and then at 4°C overnight to separate the serum. The next day, the blood was centrifuged at 2000g for 10 minutes at 4°C to aspirate the supernatant. A total of 4 serum samples were collected.

[0055] Detection of serum antibody levels in mice after immunization: In order to evaluate the humoral immune effect of mice on recombinant AEP protein, the antibody level was detected by indirect ELISA. The specific operation method is as follows: (1) Coating: Culture to OD 600The APP type 1 CVCC259 bacterial solution of about 0.6 was centrifuged at 4°C and 11000rpm for 5 minutes to collect the cells and washed three times with PBS. The cells were resuspended with PBS and ultrasonically disrupted with an ultrasonic disruptor at a frequency of 3s on and 2s off until the bacterial solution changed from turbid to clear. The protein concentration was determined by the BCA protein quantification method and adjusted to 10μg / mL. 100μL per well was coated on a 96-well plate and coated overnight at 4°C. (2) Blocking: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 minutes each time. Add 150μL of PBS containing 5% skim milk to each well and incubate at 37°C for 2 hours. (3) Incubate with primary antibody: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 minutes each time. Dilute the serum collected at different time points 800 times with PBS, add 100μL of each serum to each well in 3 replicates and add them to the plate in turn, and set up a negative control. Incubate at 37°C for 1 hour. (4) Incubate 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 1:5000 and add 100 μL per well to the plate and incubate at 37°C for 1 h. (5) Add substrate: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 min each time. Add 100 μL TMB substrate buffer to each well and react at 37°C for 30 min. (6) Stop: Add 50 μL 1M H 2 SO 4 The reaction was terminated and the OD450nm value was read on a microplate reader within 15 minutes. Figure 6 As shown, the antibody level in the AEP protein group was much higher than that in the adjuvant group and the PBS group, and there was no difference in the antibody level with the inactivated vaccine group.

[0056] 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 optical microscopy. The results of pathological section analysis were as follows: Figure 7 As shown, the lung tissue lesions in the AEP protein group were milder, similar to those in the inactivated vaccine group and the blank group; while the lung tissues in the adjuvant group and the PBS group showed inflammatory cell infiltration, hemorrhage and other lesions.

Claims

1. A recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding a recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant vector expressing the recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae according to claim 1, characterized in that: Containing the coding gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that: The recombinant vector uses pET-30a as a skeleton.

5. An engineered cell expressing the recombinant tandem epitope antigen of Actinobacillus pleuropneumoniae according to claim 1, characterized in that: Contains the coding gene according to claim 2 or the recombinant vector according to claim 3 or 4.

6. The engineered cell according to claim 5, characterized in that: The engineering cells use Escherichia coli BL21Star (DE3) as host cells.

7. The method for preparing the recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae according to claim 1, characterized in that: The method comprises the following steps: culturing and inducing the expression of the engineered cells described in claim 5 or 6, and separating and purifying the recombinant tandem epitope antigens of Actinobacillus pleuropneumoniae from the culture fluid.

8. Use of the recombinant tandem epitope antigen of porcine Actinobacillus pleuropneumoniae according to claim 1 in the preparation of porcine Actinobacillus pleuropneumoniae vaccine.

9. A porcine Actinobacillus pleuropneumoniae vaccine, characterized in that: The invention comprises the recombinant tandem epitope antigen of Actinobacillus pleuropneumoniae according to claim 1.

10. The porcine Actinobacillus pleuropneumoniae vaccine according to claim 9, characterized in that: An adjuvant is also included.

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