CN108822192B - An immunoprotective antigen protein APJL_1976 of Actinobacillus pleuropneumoniae and its application - Google Patents

Abstract

The invention discloses an immunoprotective antigen protein APJL_1976 of Actinobacillus pleuropneumoniae and its application. The amino acid sequence of the immunoprotective antigen protein of Actinobacillus pleuropneumoniae of the present invention is shown in SEQ ID NO.1, the immunoprotective antigen protein consists of 273 amino acids, and the mature polypeptide part thereof is amino acids 21-191. The nucleotide sequence encoding the immunoprotective antigen protein is preferably shown in SEQ ID NO.2. The immunoprotective antigen protein of the invention has good immunogenicity and strong immunoprotective effect, and has the application of preparing a kit for detecting Actinobacillus pleuropneumoniae antibody, and the application and preparation of porcine pleuropneumoniae subunit vaccine. Use of a medicament for the prevention of diseases caused by Actinobacillus pleuropneumoniae. The invention provides new materials for preparing porcine pleuropneumonia subunit vaccine, and has great significance for the prevention and control of porcine pleuropneumonia.

Description

A kind of Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL_1976 and its application

technical field

The invention relates to the technical field of animal infectious disease subunit vaccine preparation, in particular to an immunoprotective antigen protein APJL_1976 of Actinobacillus pleuropneumoniae and its application.

Background technique

Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) is a small gram-negative bacterium that causes porcine pleuropneumoniae. The disease is a highly contagious respiratory disease characterized by acute hemorrhagic, fibrinous, necrotizing bronchopneumonia and fibrinous pleurisy. Since it was first reported by Pattison et al in 1957, the disease has spread to many countries and regions around the world. region, seriously hindering the healthy development of the pig industry.

Vaccine immunization is an effective means to prevent and control porcine pleuropneumonia. Porcine pleuropneumoniae subunit vaccines are usually prepared by appropriate processing based on the identified immunoprotective antigenic proteins of A. Subunit vaccines are safe to use and can stimulate animals to produce high levels of antibodies against the target antigenic protein, providing animals with certain protection. However, at present, the number of immunoprotective antigen proteins that can be used to produce porcine pleuropneumoniae subunit vaccines is relatively small, which largely limits the improvement and development of porcine pleuropneumoniae subunit vaccines. Therefore, the discovery of new immunoprotective antigen proteins is of great significance for the development of high-efficiency porcine pleuropneumoniae subunit vaccines and the control of the disease.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a novel immunoprotective antigen protein APJL_1976 of Actinobacillus pleuropneumoniae. The present invention also aims to provide the application of the immunoprotective antigen protein APJL_1976.

The object of the present invention is achieved through the following technical solutions:

An Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL_1976, the amino acid sequence of which is shown in SEQ ID NO. 1, the immunoprotective antigen protein is composed of 191 amino acids, and the mature polypeptide part is amino acids 21-191. The nucleotide sequence encoding the immunoprotective antigen protein is preferably shown in SEQ ID NO.2.

A recombinant expression vector comprising the coding sequence of the immunoprotective antigen protein mature polypeptide.

A recombinant engineering bacteria, Escherichia coli comprising the recombinant expression vector.

The preparation method of the Actinobacillus pleuropneumoniae immunoprotective antigen comprises the following steps:

(1) Constructing a recombinant expression vector comprising the coding sequence of the immunoprotective antigen protein or its mature polypeptide; the backbone vector of the recombinant expression vector is preferably pGEX-KG.

(2) transforming the recombinant expression vector into a host cell; the host cell is preferably Escherichia coli;

(3) culturing the transformed host cells and inducing them to express the immunoprotective antigen; the conditions for culturing and inducing are preferably: culturing the transformed host cells at 37° C. to an OD ₆₀₀ of 0.6-0.7 , IPTG was added to a final concentration of 1.0 mM, and the culture was continued at 37°C for 3-4 hours.

(4) isolating and purifying the A. pleuropneumoniae immunoprotective antigen from the induced host cells.

The Actinobacillus pleuropneumoniae immunoprotective antigen can be combined with the rabbit anti-Acinobacter pleuropneumoniae polyclonal antibody, indicating that it has good immunoreactivity, and can be used for the detection of the antibody level of Actinobacillus pleuropneumoniae in the sample. Determination. Therefore, the above-mentioned A. pleuropneumoniae immunoprotective antigen has the application of preparing a kit for detecting A. pleuropneumoniae antibody, and the method based on the kit includes ELISA method, Western blotting method, colloidal gold immunoassay, spot Hybridization, etc.

The described Actinobacillus pleuropneumoniae immunoprotective antigen can provide effective immune protection for mice infected with Actinobacillus pleuropneumoniae after immunizing mice, indicating that it has good immunogenicity and immune protection. Therefore, the Actinobacillus pleuropneumoniae immunoprotective antigen has applications in preparing porcine pleuropneumoniae subunit vaccines, and in preparing medicines for preventing diseases caused by Actinobacillus pleuropneumoniae.

Correspondingly, the above-mentioned nucleotides encoding the immunoprotective antigens, recombinant expression vectors or recombinant engineered bacteria expressing the immunoprotective antigens also have these applications: preparation of a kit for detecting A. pleuropneumoniae antibodies Application of porcine pleuropneumoniae subunit vaccine, preparation of medicine for preventing diseases caused by Actinobacillus pleuropneumoniae.

The present invention has the following advantages and beneficial effects: the immunoprotective antigen protein APJL_1976 of the present invention has strong immunogenicity and immunoprotective effects, and mice are immunized with it and then infected with Actinobacillus pleuropneumoniae at a dose of 6×LD ₅₀ . , the survival rate was 100%; the mice passively immunized with anti-APJL_1976 hyperimmune serum were infected with Actinobacillus pleuropneumoniae at a dose of 6×LD ₅₀ , and the survival rate was over 90%. The present invention provides new material for preparing porcine pleuropneumonia subunit vaccine, and has great significance for the prevention and control of porcine pleuropneumonia.

Description of drawings

Figure 1 is a picture of the restriction enzyme digestion of the expression vector pGEX-KG-APJL_1976. Lane M: DL 15000 DNA marker; Lane 1: BamHI/HindIII digestion of plasmid pGEX-KG-APJL_1976.

Figure 2 is the SDS-PAGE detection diagram of the target protein APJL_1976 expressed by the engineered bacteria. Lane M: prestained protein marker; Lane 1: Escherichia coli/pGEX-KG empty vector whole cell lysate; Lane 2: Escherichia coli/pGEX-KG-APJL_1976 bacterial cell lysis pellet; Lane 3: Escherichia coli/pGEX-KG -APJL_1976 cell lysis supernatant; lane 4: E. coli/pGEX-KG-APJL_1976 whole cell lysate.

Figure 3 is a graph showing the results of immunoblotting analysis of the target protein. Picture A is the SDS-PAGE picture after purification of APJL_1976, and picture B is the immunoblotting picture after transmembrane transfer. Lane M: prestained protein marker; lane 1: purified GST protein; lane 2: purified APJL_1976 protein. Arrows indicate the APJL_1976 western blotting bands.

Figure 4 is a graph showing the results of the immune protection of APJL_1976 protein. Picture A shows the survival rate of mice challenged with APJL_1976 after immunization, and Picture B shows the survival rate of mice after passive immunization with anti-APJL_1976 polyclonal antibody. From 72h after infection to the end of the observation period, the survival rate of mice did not change.

Detailed ways

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The expression of embodiment 1APJL_1976 protein

1. Extraction of genomic DNA of Actinobacillus pleuropneumoniae

Take 1 mL of the bacterial liquid of Actinobacillus pleuropneumoniae (JL03) cultured overnight, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and then extract the genome of the JL03 strain according to the instructions of the genome extraction kit (Wuhan Boyue Biotechnology Co., Ltd.). The specific process is as follows: add 200 μL of RB to the cell pellet to resuspend, centrifuge at 10,000 rpm for 30 seconds, discard the supernatant, add 200 μL of RB to resuspend the pellet, then add 20 μL of lysozyme to the centrifuge tube, shake vigorously, and place it at 37°C Incubate in an incubator for 15 minutes, then add 200 μL of binding solution CB, invert and mix, add 20 μL proteinase K (20 mg/mL), mix well, put it in a 70°C water bath to react for 10 minutes, take it out and cool it and add 100 μL isozyme K (20 mg/mL). Propanol, invert and mix, then transfer all the material in the centrifuge tube to the adsorption column, centrifuge at 10,000 rpm for 30 seconds, discard the waste liquid, add 500 μL of IR, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and then add 700 μL of WB, Centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, add 500 μL of WB, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, then centrifuge at 13,000 rpm for 2 minutes, take out the adsorption column, put it into a new centrifuge tube, and add 50 μL to the middle of the adsorption membrane EB, placed at room temperature for 5 minutes, centrifuged at 12,000 rpm for 1 minute, and the resulting solution is the genome, which is stored at -20°C for later use.

2. Preparation of APJL_1976 gene

According to the nucleotide sequence of the APJL_1976 gene (as shown in SEQID NO.2 of the sequence listing), the following primers were designed:

Forward primer: 5'-TT GGATCC TGTTCGTCTTCTTCGTCTTC-3',

Reverse primer: 5'-GG AAGCTT TTATCTTACACGCAGTATTTG-3'.

The underlined part of the forward primer is the BamHI restriction site, and the underlined part of the reverse primer is the HindIII restriction site. Using the genomic DNA of Actinobacillus pleuropneumoniae JL03 as the template, PCR amplification was performed with the designed primers to obtain the coding sequence of APJL_1976 mature polypeptide. The amplification system is as follows:

PCR reaction conditions were: pre-denaturation: 94°C for 5 minutes; cycling: 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 1 minute, 30 cycles; final extension: 72°C for 10 minutes; 16°C for 2 minutes.

The fragment size and yield of PCR products were detected by 1% agarose gel, and the PCR products were purified by DNA purification kit (Wuhan Boyue Biotechnology Co., Ltd.). The specific method is as follows: After the PCR amplification products are separated by agarose gel electrophoresis, place the gel on the cutting table, turn on the UV lamp, cut the target fragment with a blade, and place it in a sterile 1.5mL centrifuge tube. Add 300 μL of sol solution, bath at 56°C for 5 minutes, shake several times during this period, and when the glue blocks are completely dissolved, transfer the solution to the adsorption column, centrifuge at 8000 rpm for 1 minute, discard the waste liquid, add 700 μL of rinse solution, and centrifuge at 8000 rpm for 1 minute , discard the waste liquid, rinse for a total of 2 times, then centrifuge at 12,000 rpm for 1 minute, put the adsorption column into a new centrifuge tube, add 30 μL of sterilized deionized water to the middle part of the adsorption membrane, 65 ℃ water bath for 2 minutes, 12,000 rpm Centrifuge for 1 minute, and the collected liquid is the purified PCR product, which is the gene fragment encoding the mature protein of APJL_1976.

3. Construction of recombinant expression vector

(1) Connect the target gene with the A/T cloning vector pMD18-T: connect the recovered PCR product to the pMD18-T vector, and the connection system is:

Recycled product 16μL pMD18-T 1μL 10×T4 DNA Buffer 2μL T4 DNA ligase 1μL

After mixing, it was sealed with parafilm, labeled as pMD18-T-APJL_1976 ligation product, and placed in a 16°C water bath for overnight connection.

(2) Transformation of ligation products and identification of plasmids

The pMD18-T-APJL_1976 ligation product was transformed into E. coli DH5α competent cells. The transformation method was as follows: take 10 μL of the ligation product and add it to E. coli DH5α competent cells. After that, take a quick ice bath for 2 minutes, then add 500 μL of LB liquid medium, incubate at 37°C for 1 hour on a shaker at 220 rpm, and then centrifuge at 7000 rpm for 3 minutes, discard 500 μL of supernatant, resuspend the cells with the remaining medium, and spread evenly on the medium containing 100 μg /mL ampicillin on LB agar plate, 37 ℃ incubator overnight. Then single colonies were picked and inoculated into LB liquid medium containing 100 μg/mL ampicillin, and cultured for 12 hours.

Extract the plasmid by alkaline lysis method: transfer the bacterial solution to a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 1 minute, and discard the supernatant. Add 100 μL of pre-cooled solution I, resuspend the cells, and place on ice; add 200 μL of solution II, invert and mix 5-6 times, ice bath for 10 minutes; add 150 μL of pre-cooled solution III, invert and mix for 5-6 times, ice bath for 10 minutes, and centrifugation at 12,000 rpm for 10 minutes. Transfer the supernatant to a new centrifuge tube, add 400 μL of isopropanol, invert and mix, place at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, add 200 μL of deionized water, dissolve the precipitate, and then add 100 μL of 7.5mol /L of ammonium acetate, invert and mix, and ice bath for 10 minutes. Centrifuge at 12000 rpm for 10 minutes. The supernatant was then transferred to a new centrifuge tube, 300 μL of isopropanol was added, mixed by inversion, and left at room temperature for 10 minutes. Then centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, add 200 μL of 75% ethanol, centrifuge at 12,000 rpm for 2 minutes, aspirate the supernatant, add 20 μL of RNase-containing water, dissolve the precipitate, and obtain a plasmid. Then it was identified by the double-enzyme digestion method, and the enzyme digestion system was as follows:

Plasmid DNA 1.0μL 10×K Buffer 1.0μL BamHI(20U/μL) 0.2μL HindIII (20U/μL) 0.2μL ddH₂O 7.6μL

After 1 hour of reaction at 37°C, stop solution was added, 5 μL of sample was taken after mixing, and detected by 1% agarose gel electrophoresis, the plasmid containing the correct inserted fragment was named pMD18-T-APJL_1976. The correctness of the target gene sequence was analyzed by bidirectional sequencing.

(3) Construction and identification of recombinant expression plasmids

The plasmid pMD18-T-APJL_1976 was digested using BamHI and HindIII, and after separation by agarose gel electrophoresis, the target fragment was recovered and connected with the prokaryotic expression plasmid pGEX-KG subjected to the same digestion treatment. The ligation reaction system is as follows:

destination fragment 12μL pGEX-KG 5μL 10×T4 DNA Buffer 2μL T4 DNA ligase 1μL

After mixing, it was sealed with parafilm, labeled as pGEX-KG-APJL_1976. After the ligation product was reacted at 16 °C overnight, the ligation product was transformed into E. coli DH5α competent cells. The transformation method, single colony picking and culture, and plasmid preparation methods were the same as above. Construction of the pMD18-T-APJL_1976 plasmid. After the plasmid pGEX-KG-APJL_1976 was double digested with BamHI and HindIII, the target fragment and the size of the vector were as expected (Fig. 1).

4. Preparation of engineering strains

The correctly identified pGEX-KG-APJL_1976 plasmid was transformed into E. coli expression strain BL21 (DE3) competent cells, coated with LB agar plates containing 100 μg/mL ampicillin, and cultured at 37 °C overnight. Pick a single colony, 37 °C After culturing for 12 hours, a recombinant bacterium containing pGEX-KG-APJL_1976 was obtained, which was an engineering bacterium. At the same time, the pGEX-KG-APJL_1976 plasmid was replaced by the pGEX-KG vector, and the steps were the same as above to obtain a recombinant bacteria containing pGEX-KG, which was used as a control.

V. Preparation and purification of APJL_1976 protein

(1) The engineered bacteria prepared in the above "four" were cultured in 100 mL of LB liquid medium containing 100 μg/mL ampicillin, and incubated at 37° C. for 3 hours. When OD ₆₀₀ = 0.6, IPTG was added to a final concentration of 1.0 mM, The incubation was continued for 3 hours at 37°C.

(2) 5000rpm centrifugation for 10 minutes to collect the thalline, resuspend the gained thalline with 20mL PBS solution, crush the thalline with a high-pressure crusher, centrifuge at 12000rpm for 10 minutes after sampling, collect the supernatant and precipitate respectively, and sample APJL_1976 by SDS-PAGE protein expression. As shown in Figure 2, SDS-PAGE electrophoresis showed that the molecular weight of APJL_1976 protein was about 53kDa (including GST tag), the molecular weight of GST control protein was about 26kDa, and APJL_1976 was mainly in the bacterial lysis supernatant. Then, the supernatant was added to the glutathione affinity chromatography column. After the binding was completed, 20 mL of PBS was added to wash the affinity chromatography column, and then 5 mL of reduced glutathione solution was added to elute the target protein. The liquid was collected in a 1.5 mL clean centrifuge tube to obtain purified recombinant APJL_1976 protein. Then add 20 mL of eluent and 20 mL of PBS to wash the chromatography column in sequence, and finally soak the chromatography column with 10 mL of 20% ethanol for preservation.

(3) The control protein GST was extracted and purified from the recombinant bacteria containing pGEX-KG according to the above method, and the purified APJL_1976 and GST proteins were detected by SDS-PAGE (Fig. 3A).

6. Western blot identification of APJL_1976 protein

The recombinant APJL_1976 protein purified in the above "five" was analyzed by immunoblotting as follows: First, a 12% polyacrylamide gel was prepared, APJL_1976 and GST proteins were spotted, and separated by SDS-PAGE electrophoresis. After electrophoresis, measure the size of the gel that needs to be transferred to the membrane, and then cut 4 equal-sized filter papers and 1 slightly larger nitrocellulose filter membrane, according to: black splint-sponge pad-two layers of filter paper-gel-filter membrane - In the order of two layers of filter paper - sponge pad - white splint, install a film transfer device, and use a glass rod to drive away air bubbles with each layer; turn on the power, and transfer at a constant pressure of 80V for 30 minutes. After the transfer, remove the nitrocellulose filter membrane and seal it with a blocking solution containing 5% skim milk at room temperature for 1 hour; take out the filter membrane and wash it three times with washing solution for 5 minutes each time; then add washing solution for 1 hour. : 400 diluted rabbit anti-A. pleuropneumoniae polyclonal antibody, incubate at 37°C for 30 minutes; remove the filter, wash 4 times with washing solution for 5 minutes each time; then add 1:5000 diluted HRP-labeled goat anti-rabbit IgG, incubate at 37°C for 30 minutes; remove the filter, wash 5 times with washing solution for 5 minutes each time; then develop color with DAB color development kit. The results showed that the APJL_1976 protein had the same color band as expected, while the GST protein had no band, as shown in Figure 3B.

Example 2 Identification of the immunoprotective properties of APJL_1976 protein

(1) Active immunization and challenge of mice

6-week-old female BALB/c mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention) were immunized with APJL_1976 protein and GST protein purified in Example 1, 12 mice in each group. At the same time, a blank control of TSB was set, and no immunization was performed. The first immunization dose was 80 μg/mouse, mixed with an equal volume of Freund's complete adjuvant, emulsified, and inoculated at multiple points subcutaneously on the back of mice. After an interval of two weeks, the second immunization was carried out at a dose of 80 μg/mice, mixed with an equal volume of incomplete Freund's adjuvant and emulsified, and subcutaneously injected into the back of the mice at multiple points. The labeled goat anti-mouse IgG was the secondary antibody (1:5000 dilution). The antibody titer of mouse anti-APJL_1976 protein reached 1:1.0×10 ⁴ . 14 days after the second immunization, the mice in each group were infected by intraperitoneal injection with a dose of 6×10 ⁶ CUF/mice (6×LD ₅₀ ) of Actinobacillus pleuropneumoniae 4074, and the mice in each group were observed for 7 days. , and recorded the morbidity and mortality of mice in each group. As shown in Figure 4A, the survival rate of the mice in the APJL_1976 immunized group was 100%, and the survival rate of the mice in the GST immunized group and the TSB blank control group was 0%.

(2) Passive immunization and challenge of mice

In the previous step of active immunization, the sera of mice in each group were collected 10 days after the secondary immunization, and mixed within the groups. Then, 6-week-old female BALB/c mice (purchased from the Experimental Animal Center of Hubei Provincial Center for Disease Control and Prevention) were inoculated by intraperitoneal injection, with 12 mice in each group, each inoculated with 50 μL for passive immunization. 3 hours after inoculation, mice in each group were infected by intraperitoneal injection with 6×10 ⁶ CUF/dose of Actinobacillus pleuropneumoniae 4074 strain, and were observed continuously for 7 days, and the mice in each group were recorded. morbidity and mortality. As shown in Figure 4B, the survival rate of mice in the anti-APJL_1976 serogroup was 92%, and the survival rate of mice in the anti-GST protein sera and TSB blank serogroups was 0%.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> Central China Normal University

<120> A kind of Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL_1976 and its application

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Met Lys Lys Ser Phe Ile Leu Phe Pro Leu Ala Val Ser Val Ser Leu

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Ala Leu Ser Gly Cys Ser Ser Ser Ser Ser Ser Ser Ser Ser Asp Gly Ala

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Glu Val Ala Asn Val Asn Ser Asn Gly Ile Ser Ser Thr Thr Ala Leu

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Pro Ser Trp Gln Ala Thr Asp Ser Ile Gln Ser Val Glu Met Pro Ala

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Ser Met Ala Ser Ala Pro Ser Arg Thr Ile Glu Gln Asn Thr Ala Tyr

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Asn Asn Gln Thr Ala Tyr Thr Gln Pro Val Asn Ser Val Pro Gln Gln

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Pro Val Val Gln Thr Pro Val Gln Ser Ala Pro Ser Tyr Thr Ser Glu

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Ser Gly Ala Gly Ser Asn Met Ile Gly Asn Cys Arg Val Val Arg Asp

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Asp Ser Ser Tyr Thr Val Gly Lys Ser Asp Thr Met Tyr Leu Ile Ser

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Tyr Leu Thr Gly Gln Thr Pro Ala Gln Ile Ala Ala Leu Asn Asn Leu

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ggtatttcat cgacaaccgc attgccgagc tggcaagcga cggattcgat tcaatcggta 180

gaaatgccgg cgtctatggc atcggctccg tcacggacaa tcgagcaaaa taccgcttat 240

aataatcaaa ccgcttatac gcaaccggtt aatagcgtac cgcaacagcc tgtggttcaa 300

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Claims (8)

1. A mature polypeptide of the immunoprotective antigen protein of Actinobacillus pleuropneumoniae, characterized in that: its amino acid sequence is amino acids 21-191 of the sequence shown in SEQ ID NO.1. 2 . A recombinant expression vector, characterized in that: it comprises the coding sequence of the mature polypeptide of claim 1 . 3. A recombinant engineering bacterium is characterized in that: it is Escherichia coli comprising the recombinant expression vector described in claim 2. 4. a preparation method of Actinobacillus pleuropneumoniae immunoprotective antigen, is characterized in that: comprise the following steps: (1) construct a recombinant expression vector comprising the mature polypeptide coding sequence of claim 1; (2) transforming the recombinant expression vector into a host cell; (3) culturing the transformed host cell and inducing it to express the immunoprotective antigen; (4) isolating and purifying the induced host cell to obtain the mature polypeptide of claim 1. 5. preparation method according to claim 4, is characterized in that: The backbone vector of the recombinant expression vector described in step (1) is pGEX-KG; The host cell described in step (2) is Escherichia coli; The conditions for culture and induction in step (3) are: culture the transformed host cells at 37°C to an OD ₆₀₀ of 0.6-0.7, add IPTG to a final concentration of 1.0 mM, and continue to culture at 37°C for 3-4 hours. 6 . The application of the mature polypeptide of claim 1 , the recombinant expression vector of claim 2 or the recombinant engineered bacteria of claim 3 in the preparation of a kit for detecting A. pleuropneumoniae antibodies. 7 . 7 . The application of the mature polypeptide of claim 1 , the recombinant expression vector of claim 2 or the recombinant engineered bacteria of claim 3 in the preparation of a porcine pleuropneumoniae subunit vaccine. 8 . 8. Use of the mature polypeptide of claim 1, the recombinant expression vector of claim 2, or the recombinant engineered bacteria of claim 3 in the preparation of a medicine for preventing diseases caused by Actinobacillus pleuropneumoniae.

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