CN108794584B

CN108794584B - Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _1380 and application thereof

Info

Publication number: CN108794584B
Application number: CN201810608304.4A
Authority: CN
Inventors: 刘金林; 祁超; 曹雨柔; 高露露; 张丽
Original assignee: Central China Normal University
Current assignee: Central China Normal University
Priority date: 2018-06-13
Filing date: 2018-06-13
Publication date: 2021-02-19
Anticipated expiration: 2038-06-13
Also published as: CN108794584A

Abstract

The invention discloses an immunoprotective antigen protein APJL_1380 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 582 amino acids, and the mature polypeptide part thereof is amino acids 25-582. 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

Actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _1380 and application thereof

Technical Field

The invention relates to the technical field of preparation of subunit vaccines for infectious diseases of animals, in particular to an actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _1380 and application thereof.

Background

Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae) is a gram-negative bacterium, small bacillus, which is a pathogenic bacterium causing porcine pleuropneumonia. The disease is a respiratory disease with high contagious degree, is mainly characterized by acute hemorrhagic, cellulosic, necrotic bronchopneumonia and cellulosic pleurisy, is spread in a plurality of countries and regions all over the world since the first report of Pattison and the like in 1957, and seriously hinders the healthy development of the pig industry.

Vaccine immunization is an effective means of preventing and controlling porcine pleuropneumonia. The porcine pleuropneumonia subunit vaccine is generally a vaccine preparation prepared by proper treatment based on identified actinobacillus pleuropneumoniae immunoprotective antigen protein. The subunit vaccine is safe to use, can stimulate animals to generate high-level antibodies against target antigen proteins, and provides certain protection for the animals. However, the number of immunoprotective antigenic proteins which can be used by people to produce porcine pleuropneumonia subunit vaccines is small, and the improvement and development of the porcine pleuropneumonia subunit vaccines are limited to a great extent. Therefore, the discovery of new immunoprotective antigen protein has important significance for developing high-efficiency porcine pleuropneumonia subunit vaccine and controlling the disease.

Disclosure of Invention

The invention aims to provide a novel actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _ 1380. The invention also aims to provide application of the immunoprotective antigen protein APJL _ 1380.

The purpose of the invention is realized by the following technical scheme:

an immunoprotective antigen protein APJL _1380 of actinobacillus pleuropneumoniae, the amino acid sequence of which is shown in SEQ ID NO.1, is composed of 582 amino acids, and the mature polypeptide part of the immunoprotective antigen protein is 25-582 amino acids. The nucleotide sequence for coding the immunoprotective antigen protein is preferably shown as SEQ ID NO. 2.

A recombinant expression vector comprising a coding sequence for said mature polypeptide of an immunoprotective antigenic protein.

A recombinant engineering bacterium, which comprises Escherichia coli of the recombinant expression vector.

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

(1) constructing a recombinant expression vector containing the immune protective antigen protein or the mature polypeptide coding sequence thereof; the skeleton 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 said transformed host cell and inducing it to express said immunoprotective antigen; the conditions for culturing and inducing are preferably as follows: the transformed host cells were cultured to OD at 37 ℃₆₀₀When the concentration is 0.6-0.7, IPTG is added to a final concentration of 1.0mM, and the mixture is transferred to 37 ℃ to continue culturing for 3-4 hours.

(4) Isolating and purifying the A. pleuropneumoniae immunoprotective antigen from the induced host cell.

The actinobacillus pleuropneumoniae immunoprotective antigen can be combined with a rabbit anti-actinobacillus pleuropneumoniae polyclonal antibody for reaction, and shows that the actinobacillus pleuropneumoniae immunoprotective antigen has good immunoreactivity and can be used for measuring the level of the actinobacillus pleuropneumoniae antibody in a sample. Therefore, the above-mentioned actinobacillus pleuropneumoniae immunoprotective antigen has an application to the preparation of a kit for detecting actinobacillus pleuropneumoniae antibodies based on methods including ELISA, western blotting, colloidal gold immunoassay, dot hybridization method, and the like.

After the actinobacillus pleuropneumoniae immunoprotective antigen is used for immunizing a mouse, effective immunoprotection can be provided for the mouse infected with the actinobacillus pleuropneumoniae, and the actinobacillus pleuropneumoniae immunoprotection antigen has good immunogenicity and immunoprotection effects. Therefore, the actinobacillus pleuropneumoniae immunoprotective antigen has the application of preparing a porcine pleuropneumonia subunit vaccine and preparing a medicament for preventing diseases caused by the actinobacillus pleuropneumoniae.

Correspondingly, the nucleotide for coding the immunoprotective antigen, the recombinant expression vector or the recombinant engineering bacterium for expressing the immunoprotective antigen also have the applications: the application of the kit for detecting the actinobacillus pleuropneumoniae antibody, the application of the subunit vaccine for porcine pleuropneumonia and the application of the medicine for preventing diseases caused by the actinobacillus pleuropneumoniae are prepared.

The invention has the following advantages and beneficial effects: the immunoprotective antigen protein APJL _1380 of the invention has strong immunogenicity and immunoprotection, and 6 XLD is used for immunizing mice after the mice are immunized by the immunoprotective antigen protein APJL _1380₅₀The survival rate of mice infected with the actinobacillus pleuropneumoniae is 100%; mice passively immunized with anti-APJL _1380 protein hyperimmune serum at 6 XLD₅₀Mice were infected with a dose of a. pleuropneumoniae, with a survival rate of 100%. The invention provides a new material for preparing the porcine pleuropneumonia subunit vaccine and has important significance for preventing and controlling the porcine pleuropneumonia.

Drawings

FIG. 1 shows the restriction enzyme identification of expression vector pGEX-KG-APJL _ 1380. Lane M: DL15000DNA marker; lane 1: plasmid pGEX-KG-APJL _1380 BamHI/HindIII.

FIG. 2 is SDS-PAGE detection diagram of engineering bacteria expressing APJL _1380 protein of interest. Lane M: pre-stained protein marker; lane 1: escherichia coli/pGEX-KG-APJL _1380 whole-cell lysate; lane 2: E.coli/pGEX-KG-APJL _1380 bacteria lysis supernatant; lane 3: E.coli/pGEX-KG-APJL _1380 thalli cracking precipitation; lane 4: escherichia coli/pGEX-KG empty vector whole-strain lysate.

FIG. 3 is a graph showing the results of immunoblot analysis of a protein of interest. Panel A shows SDS-PAGE of APJL _1380 after purification, and panel B shows immunoblotting after membrane transfer. Lane M: pre-stained protein marker; lane 1: purified GST protein; lane 2: purified APJL _1380 protein. The arrow indicates APJL _1380 protein immunoblot color development band.

FIG. 4 shows the immunoprotection results of APJL _1380 protein. The A picture is the survival rate of the mice after APJL _1380 immunization and the B picture is the survival rate of the mice after the mice passively immunized by the anti-APJL _1380 polyclonal antibody are challenged. The survival rate of mice did not change from 72h post infection to the end of the observation period.

Detailed Description

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

Example 1 expression of APJL _1380 protein

Extraction of actinobacillus pleuropneumoniae genome DNA

1mL of an overnight-cultured actinobacillus pleuropneumoniae (JL03) culture solution was centrifuged at 12000rpm for 1 minute, and the supernatant was discarded, followed by extraction of the genome of JL03 strain according to the instructions of the genome extraction kit (Wuhan Bo Yue Biotechnology Co., Ltd.). The specific process is as follows: adding 200 mu L RB into the thallus sediment for resuspension, centrifuging at 10000rpm for 30 seconds, discarding the supernatant, adding 200 mu L RB for resuspension, then adding 20 mu L lysozyme into a centrifuge tube for violent oscillation, placing in a 37 ℃ incubator for 15 minutes, then adding 200 mu L binding solution CB, reversing and mixing uniformly, adding 20 mu L proteinase K (20mg/mL), mixing uniformly, placing in a 70 ℃ water bath for reaction for 10 minutes, taking out and cooling, adding 100 mu L isopropanol, reversing and mixing uniformly, then transferring all substances in the centrifuge tube into an adsorption column, centrifuging at 10000rpm for 30 seconds, pouring the waste liquid, then adding 500 mu L IR, 12000rpm for 30 seconds, discarding the waste liquid, then adding 700 mu L WB, 12000rpm for 30 seconds, discarding the waste liquid, adding 500 mu L WB, 12000rpm for 30 seconds, discarding the waste liquid, then centrifuging at 13000rpm for 2 minutes, taking out the adsorption column, placing in a new centrifuge tube, 50 μ L of EB was added to the middle part of the adsorption membrane, and left at room temperature for 5 minutes, and centrifuged at 12000rpm for 1 minute, and the resulting solution, i.e., the genome, was stored at-20 ℃ for future use.

II, preparation of APJL _1380 Gene

According to the nucleotide sequence of the APJL _1380 gene (shown as SEQID NO.2 in the sequence table), the following primers are designed:

a forward primer: 5' -TTGGATCCTGTACCGGTACAAGTTTTTTTG-3’，

Reverse primer: 5' -GGAAGCTTTTAGTTAGCGTTTTCCACTG-3’。

The underlined portion of the forward primer is the BamHI site and the underlined portion of the reverse primer is the HindIII site. The genome DNA of actinobacillus pleuropneumoniae JL03 is used as a template, and PCR amplification is carried out by using a designed primer to obtain the APJL _1380 mature polypeptide coding sequence. The amplification system was as follows:

the PCR reaction conditions are as follows: pre-denaturation: 5 minutes at 94 ℃; and (3) circulation: 30 cycles of 94 ℃ for 30 seconds, 53 ℃ for 30 seconds, 72 ℃ for 1.5 minutes; and finally, extension: 10 minutes at 72 ℃; 16 ℃ for 2 minutes.

The fragment size and yield of the PCR product were checked with 1% agarose gel, and the PCR product was purified using DNA purification kit (Wuhan Bo Tech Biotech Co., Ltd.). The specific method comprises the following steps: after the PCR amplification product is subjected to agarose gel electrophoresis separation, the gel is placed on a gel cutting table, an ultraviolet lamp is turned on, a blade is used for cutting a target segment, the target segment is placed in a sterile 1.5mL centrifuge tube, 300 mu L of sol solution is added, water bath at 56 ℃ is carried out for 5 minutes, shaking is carried out for multiple times during the process, the gel block is completely dissolved, the solution is transferred into an adsorption column, the solution is centrifuged at 8000rpm for 1 minute, waste liquid is poured out, 700 mu L of rinsing liquid is added, the solution is centrifuged at 8000rpm for 1 minute, the waste liquid is discarded, the rinsing is carried out for 2 times in total, then, the solution is centrifuged at 12000rpm for 1 minute, the adsorption column is placed into a new centrifuge tube, 30 mu L of sterilized deionized water is added into the middle part of an adsorption membrane, the solution is centrifuged at 65 ℃ for 2 minutes and 12000rpm for 1 minute, and liquid is collected.

Construction of recombinant expression vector

(1) Connecting the target gene with an A/T cloning vector pMD 18-T: the recovered PCR product was ligated into pMD18-T vector as follows:

recovering the product	16μL
		pMD18-T	1μL
10×T4DNA Buffer	2μL
		T4DNA ligase	1μL

After mixing, the mixture was sealed with a sealing film, labeled as pMD18-T-APJL _1380 ligation product, and ligated overnight in a 16 ℃ water bath.

(2) Transformation of ligation products and identification of plasmids

pMD18-T-APJL _1380 ligation product was transformed into E.coli DH5 α competent cells by: mu.L of the ligation product was added to E.coli DH 5. alpha. competent cells, mixed and incubated in ice for 20 minutes, heat-shocked in water bath at 42 ℃ for 90 seconds, rapidly incubated in ice for 2 minutes, then added with 500. mu.L of LB liquid medium, shake-cultured at 37 ℃ and 220rpm for 1 hour, centrifuged at 7000rpm for 3 minutes, the supernatant 500. mu.L was discarded, the cells were resuspended in the remaining medium, spread evenly on LB agar plates containing 100. mu.g/mL ampicillin, and incubated overnight at 37 ℃ in an incubator. Then, a single colony was picked up and inoculated into LB liquid medium containing 100. mu.g/mL ampicillin, and cultured for 12 hours.

Plasmid extraction by alkaline lysis: the bacterial liquid was transferred to a 1.5mL centrifuge tube, centrifuged at 12000rpm for 1 minute, and the supernatant was discarded. Adding 100 mu L of precooled solution I, resuspending the thalli, and placing on ice; adding 200 mu L of solution II, reversing and uniformly mixing for 5-6 times, and carrying out ice bath for 10 minutes; add 150. mu.L of pre-cooled solution III, mix 5-6 times by inversion, ice-wash for 10 min, and centrifuge at 12000rpm for 10 min. Transferring the supernatant to a new centrifuge tube, adding 400 mu L of isopropanol, reversing and mixing uniformly, standing at room temperature for 10 minutes, centrifuging at 12000rpm for 10 minutes, discarding the supernatant, adding 200 mu L of deionized water, dissolving the precipitate, adding 100 mu L of 7.5mol/L ammonium acetate, reversing and mixing uniformly, and carrying out ice bath for 10 minutes. Centrifuge at 12000rpm for 10 minutes. The supernatant was then transferred to a new centrifuge tube, 300. mu.L of isopropanol was added, mixed by inversion, and left at room temperature for 10 minutes. Then, the mixture was centrifuged at 12000rpm for 10 minutes, the supernatant was discarded, 200. mu.L of 75% ethanol was added, and the mixture was centrifuged at 12000rpm for 2 minutes, and the supernatant was aspirated and removed, and 20. mu.L of RNase-containing water was added to dissolve the precipitate, thereby obtaining a plasmid. Then, the enzyme digestion system is identified by a double enzyme digestion method 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 ℃, stop solution was added, 5. mu.L of the sample was collected after mixing, and the plasmid containing the correct insert was named pMD18-T-APJL _1380 by 1% agarose gel electrophoresis. The correctness of the target gene sequence is analyzed by bidirectional sequencing.

(3) Construction and identification of recombinant expression plasmids

BamHI and HindIII double enzyme digestion plasmid pMD18-T-APJL _1380 is used, after agarose gel electrophoresis separation, a target fragment is recovered and is connected with prokaryotic expression plasmid pGEX-KG which is subjected to the same enzyme digestion treatment, and a connection reaction system is as follows:

segment of interest	12μL
		pGEX-KG	5μL
10×T4DNA Buffer	2μL
		T4DNA ligase	1μL

After mixing, sealing with a sealing film, marking as pGEX-KG-APJL _1380, reacting the ligation product at 16 ℃ overnight, transforming the ligation product to escherichia coli DH5 alpha competent cells, and constructing the pMD18-T-APJL _1380 plasmid through the transformation method, single colony picking culture and plasmid preparation method. After double digestion of plasmid pGEX-KG-APJL _1380 with BamHI and HindIII, the desired fragment and vector size were obtained in accordance with expectations (FIG. 1).

Preparation of engineering strain

The correctly identified pGEX-KG-APJL _1380 plasmid is transformed into a competent cell of an escherichia coli expression strain BL21(DE3), an LB agar plate containing 100 mu g/mL ampicillin is coated, after overnight culture at 37 ℃, a single colony is selected and cultured for 12 hours at 37 ℃, and the recombinant bacterium containing pGEX-KG-APJL _1380, namely the engineering bacterium, is obtained. Meanwhile, pGEX-KG vector was used instead of pGEX-KG-APJL _1380 plasmid, and the procedure was as above, to obtain recombinant bacteria containing pGEX-KG as a control.

Preparation and purification of APJL _1380 protein

(1) The engineered bacteria prepared in the above "four" were cultured in 100mL of LB liquid medium containing 100. mu.g/mL ampicillin, and cultured at 37 ℃ for 3 hours to OD₆₀₀When the concentration was 0.6, IPTG was added to a final concentration of 1.0mM, and the culture was continued at 37 ℃ for 3 hours.

(2) The cells were collected by centrifugation at 5000rpm for 10 minutes, the obtained cells were resuspended in 20mL of PBS solution, disrupted by a high pressure disruptor, and then centrifuged at 12000rpm for 10 minutes after sampling, and the supernatant and the precipitate were collected, respectively, and the expression of APJL _1380 protein was detected by SDS-PAGE after sampling. As shown in FIG. 2, SDS-PAGE showed that APJL _1380 protein had a molecular weight of about 53kDa (including GST tag), GST control protein had a molecular weight of about 26kDa, and APJL _1380 was mainly in bacterial lysis supernatant. And then adding the supernatant into a glutathione affinity chromatography column, after the binding is finished, adding 20mL of PBS to wash the affinity chromatography column, then adding 5mL of reduced glutathione solution to elute the target protein, and collecting the eluent into a 1.5mL clean centrifugal tube to obtain the purified recombinant APJL _1380 protein. Then 20mL of eluent and 20mL of PBS are added in sequence to wash the chromatographic column, and finally the chromatographic column is soaked and preserved by 10mL of 20% ethanol.

(3) GST, control proteins were extracted and purified from recombinant bacteria containing pGEX-KG as described above, and the purified APJL _1380 and GST proteins were detected by SDS-PAGE (FIG. 3A).

Sixth, APJL _1380 protein immunoblotting identification

Immunoblot analysis of recombinant APJL _1380 protein purified in "five" above was performed as follows: first, a 12% polyacrylamide gel was prepared, and APJL _1380 and GST proteins were spotted and separated by SDS-PAGE. After electrophoresis, the gel size of the membrane needs to be changed in the measurement, and then 4 pieces of filter paper with the same size and 1 piece of nitrocellulose filter membrane with a larger size are cut according to the following steps: mounting a film rotating device in the sequence of black splint-spongy cushion-two layers of filter paper-gel-filter membrane-two layers of filter paper-spongy cushion-white splint, and removing bubbles by using a glass rod when one layer is laid; the power is turned on, and the 80V constant voltage transfer is carried out for 30 minutes. After the transfer printing is finished, taking down the cellulose nitrate filter membrane, and sealing for 1 hour at normal temperature by using sealing liquid containing 5% of skim milk; taking out the filter membrane, and washing with washing solution for 3 times, 5 minutes each time; then adding a rabbit anti-actinobacillus pleuropneumoniae polyclonal antibody diluted by washing liquid at a ratio of 1:400, and incubating for 30 minutes at 37 ℃; taking out the filter membrane, and washing with washing solution for 4 times, 5 minutes each time; adding HRP-labeled goat anti-rabbit IgG diluted at the ratio of 1:5000, and incubating for 30 minutes at 37 ℃; taking out the filter membrane, and washing with washing solution for 5 times, each time for 5 minutes; then, the color is developed by using a DAB color development kit. The results show that the APJL _1380 protein lane shows a color band consistent with the expected one, while the GST protein does not show a band, as shown in FIG. 3B.

Example 2 identification of the immunoprotection of the APJL _1380 protein

(1) Active immunization and challenge of mice

Female BALB/c mice (purchased from Experimental animals center in the Hubei province disease prevention and control center) at 6 weeks of age were immunized with APJL _1380 protein and GST protein purified in example 1, respectively, and 12 mice were each group. TSB blank control was also set and no immunization was performed. The first immunization dose is 80 mug/mouse, and the first immunization dose is mixed and emulsified with equal volume of Freund complete adjuvant, and is inoculated on the back of the mouse by subcutaneous multi-point injection. Two weeks later, a second immunization was performed at a dose of 80. mu.g/mouse, mixed and emulsified with an equal volume of Freund's incomplete adjuvant, and inoculated subcutaneously in the back of the mice at multiple points, and after 10 days, the antibody level was measured by ELISA using HRP-labeled goat anti-mouse IgG as a secondary antibody (1:5000 dilution). The antibody titer of the mouse anti-APJL _1380 protein reaches 1:3.8 multiplied by 10⁴. The toxicity test is carried out 14 days after the second immunization, and the toxicity test is carried out at 6 multiplied by 10⁶CUF/only (6 × LD)₅₀) The mice of each group were infected with the serum type 1 actinobacillus pleuropneumoniae 4074 strain by intraperitoneal injection, and the mice of each group were continuously observed for 7 days, and the morbidity and mortality of the mice of each group were recorded. As shown in FIG. 4A, the survival rate of APJL _1380 mice in the immunization group was 100%, and the survival rate of GST immunization group and TSB blank control group was 0%。

(2) Passive immunization and challenge of mice

In the previous active immunization, the serum of mice 10 days after the secondary immunization of each group was collected and mixed in the group. Then, female BALB/c mice (purchased from the experimental animal center of Hubei province disease prevention and control center) with the age of 6 weeks were respectively inoculated with 12 mice per group, and each animal was inoculated with 50. mu.L of the mice for passive immunization. 3 hours after inoculation, by intraperitoneal injection at 6X 10⁶Each group of mice was infected with CUF/serum type 1 actinobacillus pleuropneumoniae 4074 strain by intraperitoneal injection, and the mice were continuously observed for 7 days, and the morbidity and mortality of each group of mice were recorded. As shown in fig. 4B, the survival rate of mice in immune serogroup against APJL _1380 protein was 100%, and the survival rate of mice in immune serogroup against GST protein and TSB blank serogroup was 0%.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of Master in China

<120> actinobacillus pleuropneumoniae immunoprotective antigen protein APJL _1380 and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 582

<212> PRT

<213> Actinobacillus pleuropneumoniae

<400> 1

Met Ala Thr Ile Leu Lys Gln Lys Ile Lys Thr Val Phe Val Pro Thr

1 5 10 15

Ala Met Ala Leu Phe Leu Ser Ala Cys Thr Gly Thr Ser Phe Phe Glu

20 25 30

Asn Pro Leu Thr Lys Thr Val Lys Asp Glu Ala Tyr Ala Thr Ser Glu

35 40 45

Phe Tyr Ile Asn Lys Ala Asp Arg Ala Thr Asp Lys Glu Asp Lys Ile

50 55 60

Thr Tyr Arg Leu Leu Ala Val Arg Lys Leu Ile Asp Glu Asn Lys Ala

65 70 75 80

Ala Glu Ala Gln Asn Thr Phe Asp Asp Leu Thr Leu Ser Leu Ala Asp

85 90 95

Ile Gln Lys Asn Glu Ile Gln Lys Val Glu Tyr Asn Leu Val Ala Ala

100 105 110

Gln Leu Ala Ala Leu Gln Gly Asn Glu Ala Gln Ala Val Ser Leu Leu

115 120 125

Arg Leu Val Pro Thr Thr Gln Leu Ser Arg Thr Gln Ser Met Arg Tyr

130 135 140

Tyr Gln Thr Gln Ala Arg Ile Ala Glu Asn Arg Lys Asp Val Leu Glu

145 150 155 160

Ala Val Arg Ala Arg Ser Leu Met Thr Ser Gln Leu Ile Asp Asn Lys

165 170 175

Leu Arg Gln Glu Asn Asn Asn Gln Ile Trp Ser Leu Leu Arg Asn Ala

180 185 190

Asn Lys Gly Ala Leu Ser Ile Ala Asn Pro Gly Pro Gly Glu Thr Glu

195 200 205

Phe Ala Gly Trp Leu Ala Leu Ile Ala Val Tyr Asn Gln Asn Val Ser

210 215 220

Thr Pro Ala Gln Met Pro Gln Gly Ile Asn Asn Trp Lys Gln Leu Tyr

225 230 235 240

Pro Asn His Ser Ala Ile Thr Val Met Pro Ala Glu Leu Gln Asn Val

245 250 255

Ser Asn Phe Gln Gln Thr Gln Leu Asn Gly Val Ala Leu Leu Leu Pro

260 265 270

Leu Ser Gly Asp Ala Lys Ile Leu Gly Asp Ile Ile Lys Lys Gly Phe

275 280 285

Asn Asp Ala Lys Gly Ala Asp Ser Ile Pro Val Gln Thr Tyr Asp Thr

290 295 300

Asp Ser Gly Ser Val Glu Ser Ile Leu Ala Gln Ala Lys Gln Gln Gly

305 310 315 320

Ala Gln Thr Ile Ile Gly Pro Leu Leu Lys Ser Arg Val Asp Glu Met

325 330 335

Leu Leu Ser Pro Glu Ile Arg Asn Val Asn Val Leu Ala Leu Asn Ser

340 345 350

Thr Pro Asn Val Lys Ala Ile Pro Gly Val Cys Tyr Tyr Gly Leu Ser

355 360 365

Pro Glu Ala Glu Ala Arg Ala Gly Ala Asp Arg Leu Tyr Arg Asp Gly

370 375 380

Tyr Ser Arg Ala Ile Val Ala Ala Ser Gln Asp Asp Phe Gly Gln Arg

385 390 395 400

Ser Ala Asp Ala Phe Ser Gln Arg Trp Arg Gln Leu Thr Asn Thr Asp

405 410 415

Ala Asp Val Arg Tyr Tyr Asn Ile Pro Gln Asp Ala Val Val Ala Ile

420 425 430

Gln Asn Ser Gly Gly Val Gln Gly Ala Ala Leu Tyr Ala Leu Gly Thr

435 440 445

Ala Glu Gln Leu Leu Glu Leu Lys Gln Gly Ile Asp Gly Ser Ser Leu

450 455 460

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

465 470 475 480

Asn Gly Ile Glu Phe Arg Thr Ala Met Glu Gly Val Lys Phe Ser Glu

485 490 495

Ile Pro Leu Leu Ala Asp Ser Asn Ser Asp Glu Tyr Lys Lys Ala Glu

500 505 510

Thr Leu Ala Glu Ser Asp Phe Ser Met Met Arg Leu Tyr Ala Met Gly

515 520 525

Ser Asp Ala Trp Ala Leu Ala Asn Lys Phe Asn Glu Phe Arg Gln Ile

530 535 540

Pro Gly Tyr Ser Val Ser Gly Leu Thr Gly Asn Leu Thr Ala Ser Pro

545 550 555 560

Asn Cys Asn Ile Glu Arg Gly Met Ser Trp Leu Gln Tyr Arg Asn Gly

565 570 575

Ala Val Glu Asn Ala Asn

580

<210> 2

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<213> Actinobacillus pleuropneumoniae

<400> 2

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tttctttctg cttgtaccgg tacaagtttt tttgaaaacc cattaaccaa aacggtcaaa 120

gacgaagcat acgcaacgtc ggagttttat atcaataaag cggatcgagc cacagataaa 180

gaagataaaa ttacttatcg cctattagcc gttcgtaaac tgattgatga aaataaagcg 240

gcagaagcac aaaatacctt cgacgatctc actctatcgc tcgccgatat ccagaaaaat 300

gaaattcaaa aagtagaata taacttggtt gcggcgcaac ttgcggcatt acagggtaat 360

gaagcacaag ccgtttcact tttaagactg gtgccgacga cccaattaag ccgtacgcaa 420

agtatgcgtt actatcaaac acaagcgcgt attgcggaaa atcgtaaaga cgtgcttgaa 480

gcggtgagag cgcgttcttt aatgacttct caattaatcg ataataagtt acgccaagaa 540

aataacaatc aaatttggtc attattacgt aatgcgaata aaggtgcgtt gtctattgcc 600

aatccgggac cgggcgaaac cgagtttgcc ggttggttag ctttaattgc ggtttacaac 660

caaaacgttt cgacaccggc acaaatgccg cagggtatta ataactggaa acaactctat 720

ccgaatcata gtgcgataac ggtcatgccg gcggagttac aaaacgtatc gaatttccaa 780

caaacccaat taaacggcgt cgctttactt ttaccgttaa gcggtgacgc taaaatttta 840

ggcgatatta tcaaaaaagg ctttaatgac gctaaaggcg cggactccat tccggtgcaa 900

acatacgata cagatagcgg ttcggtcgaa agtattttgg cacaggctaa gcaacaaggc 960

gcacaaacga ttatcggtcc gttactaaaa tctcgtgtcg atgaaatgtt gttaagtcct 1020

gaaatccgaa atgtgaatgt attggcttta aattcgacgc caaatgtaaa agcgattccg 1080

ggcgtatgtt attacggttt atcgccggaa gcggaagcga gagccggagc ggatcgctta 1140

tatcgcgacg gttattcgcg tgctatcgtt gccgcatcac aagatgattt cggacaacgt 1200

tccgcagatg cattctcaca acgttggcga caattaacca atacggatgc ggacgtacgc 1260

tattacaata ttcctcaaga tgcggttgtc gcaattcaga attcgggcgg tgtgcaaggt 1320

gcggcattat atgcgttagg taccgccgag cagttacttg aattaaaaca aggtatcgac 1380

ggttcatcgc ttgccggaca attgaatatt tatacctctt cacgcagtaa ttcgccgaat 1440

aacggtatcg aattccgtac cgcaatggaa ggagtgaaat tcagtgaaat tcctctgctt 1500

gcggactcga attcggacga atataaaaaa gcggaaactt tagcggaaag tgatttctcg 1560

atgatgcgtt tatatgcaat gggttccgat gcttgggcgt tagcaaataa attcaatgaa 1620

ttccgccaaa ttccgggtta tagcgtttca ggtttaaccg gtaatttaac cgccagtcct 1680

aactgtaata tcgaacgcgg aatgtcttgg ttgcaatatc gtaatggcgc agtggaaaac 1740

gctaactaa 1749

<210> 3

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttggatcctg taccggtaca agtttttttg 30

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggaagctttt agttagcgtt ttccactg 28

Claims

1. the application of a mature polypeptide of Actinobacillus pleuropneumoniae immunoprotective antigen protein in the preparation of porcine pleuropneumoniae subunit vaccine, it is characterized in that: the aminoacid sequence of described mature polypeptide is the sequence shown in SEQ ID NO.1 of amino acids 25-582.

2 . The application of a recombinant expression vector in the preparation of a porcine pleuropneumoniae subunit vaccine, wherein the recombinant expression vector comprises the mature polypeptide described in claim 1 .

3. the application of a recombinant engineered bacteria in the preparation of porcine pleuropneumoniae subunit vaccine, characterized in that: the recombinant engineered bacteria are Escherichia coli comprising the recombinant expression vector described in claim 2.

4. the application of a mature polypeptide of Actinobacillus pleuropneumoniae immunoprotective antigen protein in the preparation of the medicine for preventing the disease caused by Actinobacillus pleuropneumoniae, it is characterized in that: the amino acid sequence of described mature polypeptide is Amino acids 25-582 of the sequence shown in SEQID NO.1.

5 . The application of a recombinant expression vector in the preparation of a medicine for preventing diseases caused by Actinobacillus pleuropneumoniae, wherein the recombinant expression vector comprises the mature polypeptide described in claim 1 .

6. the application of a recombinant engineered bacteria in the preparation of the medicine for preventing the disease caused by Actinobacillus pleuropneumoniae, it is characterized in that: described recombinant engineered bacteria is the large intestine comprising the described recombinant expression vector of claim 2 Bacillus.