CN103555645B - Resistance marker-free attenuated live vaccine against porcine contagious pleuropneumonia (PCP) and application thereof - Google Patents

Abstract

The invention discloses a porcine infectious pleuropneumonia attenuated live vaccine without a resistance marker and its application, and also discloses a strain of Actinobacillus pleuropneumoniae serotype 7 without a resistance marker for preparing the vaccine The three-gene deletion strain belongs to the technical field of bacterial genetic engineering. The Actinobacillus pleuropneumoniae serotype 7 three-gene deletion strain APPΔclpPΔapxⅡCΔfur without resistance markers disclosed by the present invention adopts directional homologous recombination technology to synthesize the proteases ClpP and ApxⅡ in Actinobacillus pleuropneumoniae (APP) The strains obtained by inactivating the genes encoding the toxin activator ApxⅡC and the iron uptake regulatory protein Fur destroyed the expression of ClpP, ApxⅡC and Fur proteins. The three-gene deletion strain obtained by the present invention is less virulent than the parent strain, and the virulence is reduced by more than 100 times, which is safe for animals; after immunizing pigs, it can provide good protection against the attack of virulent strains with different serotypes of APP, and the protection rate is 80%. Above, it can be used as attenuated live vaccine for the immune prevention of porcine infectious pleuropneumonia.

Description

A kind of attenuated live vaccine against porcine infectious pleuropneumonia without resistance marker and its application

technical field

The invention relates to a porcine infectious pleuropneumonia attenuated live vaccine without resistance markers and its application, in particular to an Actinobacillus pleuropneumoniae gene deletion strain comprising ClpP protease, ApxⅡC and Fur without resistance markers The prepared attenuated live porcine infectious pleuropneumonia vaccine and its application in preventing and treating porcine infectious pleuropneumonia belong to the technical field of bacterial genetic engineering.

Background technique

Porcine contagious pleuropneumonia (PCP) is a highly contagious and fatal respiratory infectious disease caused by Actinobacillus pleuropneumoniae (APP). At present, the disease has been widely prevalent in countries all over the world, causing huge economic losses, and has become one of the internationally recognized important infectious diseases that endanger the modern pig industry. With the large-scale and intensive development of modern pig breeding in my country, the occurrence of this disease is in an explosive trend, and the positive rate of some pig farms has reached more than 70%, which seriously hinders the healthy development of pig farming in my country.

According to the antigenicity of APP surface capsule (CPS) and lipopolysaccharide (LPS), APP can be divided into 15 serotypes, serotype 1 is further divided into two subtypes 1a and 1b, and serotype 5 is further divided into 5a and 5b two subtypes. At present, serotypes 1, 2, 3, 4, 5, 7, 8 and 15 have been discovered and isolated in my country, and the main prevalent serotypes are 1, 3, 5 and 7.

Apx toxin is the main virulence factor of APP. Four Apx toxins, ApxⅠ, ApxⅡ, ApxⅢ and ApxⅣ, have been found in all 15 serotypes of APP. Different serotypes of APP produce different Apx toxins, among which serotype 7 contains ApxⅡ and ApxⅣ.

The typical structure of the Apx toxin operon contains a complete CABD gene; among them, the A gene encodes the Apx toxin structural protein, which has no toxin activity at the beginning of synthesis, and the C gene encodes the Apx toxin activating protein, which is responsible for acetylating the structural protein to form ApxC- ApxA complex, the toxin activity ensues. The encoded proteins of gene B and gene D form a transmembrane channel responsible for the transport and secretion of Apx toxin. The ApxI and ApxIII operons have complete CABD genes, while the ApxII operon lacks the B and D genes, and its products are transported and secreted outside the cell by the BD gene-encoded product of ApxI. The deleted ApxIIC of the present invention is the activation protein of the ApxIIA toxin, and the ApxII toxin will lose its toxicity after the gene is deleted, and the toxicity of the mutant strain is greatly reduced.

Biofilm (Biofilm) is a fixed bacterial community formed by bacteria and their polysaccharide-protein complexes that accumulate and grow on the surface of organisms and non-organisms. It is an important cause of persistent infection, drug resistance and immune escape of pathogenic bacteria. one. Long-term latency of APP in pigs often causes persistent infection. Many clinical isolates obtained in recent years have multi-drug resistance and can generally form biofilms, which are considered to play an important role in the pathogenic process of APP.

Biofilm production is a highly complex and dynamic process regulated by a variety of genes involved in bacterial adhesion, metabolism, quorum sensing, and stress response. In recent years, studies on the stress response of pathogenic bacteria and infectious diseases have found that ClpP protease, a stress protein, is a very important virulence factor. It is an ATP-dependent serine protease that regulates the adaptation of bacteria to various environmental stresses. (such as heat shock, nutritional deficiency, oxidative stress, etc.), maintain the morphology and virulence of bacteria, and protect them in various environments. Studies have found that the insertion mutation or deletion of this gene usually reduces the tolerance of bacteria to adverse factors, destroys the growth of the bacteria, reduces the viability, and affects the formation of biofilm, resulting in weakened virulence. The invention deletes the ClpP protease gene to weaken the toxicity of the mutant strain.

Iron is an essential element for the growth of bacteria. Usually, because there are few free Fe ions in the animal body, it cannot meet the needs of bacterial growth. In order to adapt to the living conditions of this low Fe ion concentration in the body, bacteria have produced various mechanisms to take up enough iron. Among them, the iron absorption regulatory protein Fur is an important regulatory protein that affects bacterial iron absorption. It regulates the expression of many transferrins, which in turn affects the survival and pathogenicity of bacteria. It is an important virulence factor. The study also found that insertional mutations or deletions of this gene generally reduce bacterial colonization in host animals. Although only a few pathogenic bacteria Fur protein functions have been explored, it has shown extremely important research value and vaccine application prospects in revealing the pathogenic mechanism and vaccine research. In the present invention, the Fur regulatory protein gene is deleted, so that the virulence of the mutant strain is further weakened.

The currently used whole-bacteria inactivated vaccines and subunit vaccines can alleviate the clinical symptoms and mortality caused by homologous serotype infection, but they cannot prevent lung lesions and chronic infection, and cannot provide good treatment for heterologous serotype infection. cross-protection against any heterologous serotype, whereas natural or experimental infection can induce protection against any heterologous serotype. Therefore, the research and development of highly effective attenuated vaccines will be a feasible method to solve the shortage of current pleuropneumonia vaccines.

In view of the above background, the popular APP serotype 7 clpP, apxⅡC and fur gene deletion strains in my country were constructed, and their biological characteristics such as genetic characteristics, growth characteristics, virulence, safety to animals and protective effect were studied, and the infectivity of pigs was determined. It lays an important foundation for the prevention and treatment of pleuropneumonia and vaccine research for other respiratory infectious diseases.

Contents of the invention

One of the objectives of the present invention is to obtain an Actinobacillus pleuropneumoniae clpP, apxIIC and fur gene deletion strain without resistance marker.

The present invention provides a three-gene deletion strain of Actinobacillus pleuropneumoniae that does not contain a resistance marker. Deletion mutant strains obtained after survival.

In the present invention, preferably, the Actinobacillus pleuropneumoniae is APP serotype 7 strain, more preferably APP serotype 7 CVCC265 strain.

In the present invention, preferably, the amino acid sequence of the protease ClpP is shown in SEQ ID NO.2, the amino acid sequence of the ApxII toxin activator ApxIIC is shown in SEQ ID NO.4, and the iron absorption regulatory protein Fur The amino acid sequence is shown in SEQ ID NO.6.

In the present invention, preferably, the gene-deleted strain is APPΔclpPΔapxⅡCΔfur, which is classified as Actinobacillus pleuropneumoniae (APP), and is preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures, with an address in Chaoyang, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 1 Hospital, Beichen West Road, District, the strain preservation number is: CGMCC No.7930, and the preservation time is July 16, 2013.

The second object of the present invention is to provide a method for constructing the three-gene deletion strain of Actinobacillus pleuropneumoniae, which is characterized in that the deletion strain is obtained by introducing DNA fragments ΔclpP, ΔapxⅡC and Δfur into the Actinobacillus pleuropneumoniae Bacillus serotype 7 strain achieved by homologous recombination;

Wherein, the DNA fragment ΔclpP is followed by the upper homology arm clpPS and the lower homology arm clpPX from upstream to downstream: the upper homology arm clpPS and the lower homology arm clpPX can be combined with the Actinobacillus pleuropneumoniae Homologous recombination occurs between the upstream sequence and the downstream sequence of the ClpP protease coding gene, and the coding gene of the ClpP protease is inactivated to obtain the clpP single gene deletion strain APPΔclpP;

Wherein, the DNA fragment ΔapxⅡC is the upper homology arm apxⅡCS and the lower homology arm apxⅡCX from upstream to downstream, and the upper homology arm apxⅡCS and the lower homology arm apxⅡCX can be combined with the above-mentioned Actinobacillus pleuropneumoniae Homologous recombination occurs between the upstream sequence and the downstream sequence of the ApxⅡC protein coding gene to inactivate the coding gene of the ApxⅡC protein, and further obtain the clpP and apxⅡC double gene deletion strain APPΔclpPΔapxⅡC;

Wherein, the DNA fragment Δfur is followed by an upper homology arm furS and a lower homology arm furX from upstream to downstream, and the upper homology arm furS and the lower homology arm furX can be combined with the A. Homologous recombination occurs between the upstream sequence and the downstream sequence of the Fur protein coding gene to inactivate the Fur protein coding gene, and further obtain the clpP, apxⅡC and fur three gene deletion strain APPΔclpPΔapxⅡCΔfur.

In a specific embodiment of the present invention, the coding gene of the ClpP protease is shown in SEQ ID NO.1, the coding gene of the ApxⅡC protein is shown in SEQ ID NO.3, and the coding gene of the Fur protein As shown in SEQ ID NO.5.

The third object of the present invention is to provide the application of the three-gene deleted strain of Actinobacillus pleuropneumoniae in the preparation of attenuated live porcine infectious pleuropneumonia vaccine.

The fourth object of the present invention is to provide a live attenuated porcine infectious pleuropneumonia vaccine, the active ingredient of which is the triple-gene-deleted strain of Actinobacillus pleuropneumoniae described in the present invention.

In a specific embodiment of the present invention, a kind of Actinobacillus pleuropneumoniae three-gene deletion strain without resistance marker of the present invention is realized through the following technical scheme:

The present invention does not contain the resistance marker Actinobacillus pleuropneumoniae serum type 7 clpP, apxⅡC and fur gene deletion mutant strain APPΔclpPΔapxⅡCΔfur, which is derived from the Actinobacillus pleuropneumoniae CVCC265 strain purchased by China Veterinary Drug Administration. The clpP gene on the genome was deleted by 491 bp, and the clpP gene was inactivated to prevent the expression of ClpP protease, and a single gene deletion mutant strain was obtained; then the apxⅡC gene on the genome was deleted by 270 bp, the apxⅡC gene was inactivated, so that the ApxⅡC protein was not expressed, A double-gene deletion mutant strain was obtained; then the fur gene on the genome was deleted by 349 bp, the fur gene was inactivated, and the Fur protein was not expressed, and a three-gene deletion mutant strain was obtained.

Basic construction method of the present invention is:

1. First, using Actinobacillus pleuropneumoniae serotype 7 (abbreviated APP7, the same below) CVCC265 strain as the starting material, the upper homologous arm ClpPS and the same below of the clpP gene were amplified from the genome of CVCC265 by PCR For the source arm ClpPX, the upper and lower homology arms of the clpP gene were spliced by the overlap extension PCR method to amplify the ΔclpP gene fragment, which lacked the 491bp sequence fragment of the clpP gene. Then the modified ΔclpP sequence was cloned into the vector pUC18, and finally became the homologous recombination vector (suicide plasmid) pUCΔclpP. The suicide plasmid pUCΔclpP is transformed into the APP7CVCC265 strain by electroporation. Since the homologous recombination vector (suicide plasmid) cannot replicate autonomously in the APP bacteria, the supercoiled suicide plasmid transferred into the cells of the recipient bacteria will be is linearized. Because the suicide plasmid contains the homologous sequence of the recipient bacterium gene, it will be inserted into the chromosome of the recipient bacterium CVCC265, and then the linearized plasmid will be replaced with the clpP gene, which is the so-called single exchange homologous recombination event. occur. Only the linearized suicide plasmid replaced on the CVCC265 chromosome can exist with the replication of the chromosome. The recipient bacteria were spread on the TSA plate containing the selection marker, and the single exchange was screened by PCR. Since the single exchange is the replacement of the entire carrier sequence (including the screening drug resistance gene) on the chromosome, we must further screen for the double exchange in which the target gene is integrated into the chromosome and the carrier sequence is deleted at the same time. The homologous recombination single exchange obtained was cultured in liquid, spread on a non-resistant TSA plate, and a single colony was picked for PCR screening of positive homologous recombination double exchange, that is, the clpP single gene deletion strain APPΔclpP.

2. Then, the upper homology arm apxⅡCS and the lower homology arm apxⅡCX of the apxⅡC gene were amplified from the genome of CVCC265 by PCR, and the upper and lower homology arms of the apxⅡC gene were spliced by overlapping extension PCR method to amplify ΔapxⅡC Gene fragment, this fragment is missing the 270bp sequence fragment of apxⅡC gene. Then clone the modified ΔapxⅡC sequence into the vector pUC18, and finally become the homologous recombination vector (suicide plasmid) pUCΔapxⅡC, and then follow the method of 1 to obtain the clpP single gene deletion strain APPΔclpP as the starting material, and further delete the apxⅡC gene , to obtain clpP and apxⅡC double gene deletion strain APPΔclpPΔapxⅡC.

3. Finally, the upper homology arm furS and the lower homology arm furX of the fur gene were amplified from the genome of CVCC265 by PCR, and the upper and lower homology arms of the fur gene were spliced by overlapping extension PCR to amplify Δfur Gene fragment, this fragment is missing the 349bp sequence fragment of fur gene. Then clone this modified Δfur sequence into the vector pUC18, and finally become the homologous recombination vector (suicide plasmid) pUCΔfur, and then follow the method in 1 to obtain the clpP and apxⅡC double gene deletion strain APPΔclpPΔapxⅡC as the starting material, and further delete The fur gene was used to obtain the deletion strain APPΔclpPΔapxⅡCΔfur of clpP, apxⅡC and fur.

The Actinobacillus pleuropneumoniae three-gene deletion strain APPΔclpPΔapxIICΔfur provided by the invention has a virulence reduction of more than 100 times compared with the parent strain CVCC265 strain, and has good safety to test mice and pigs. After the animals were immunized with the three-gene-deleted strain of Actinobacillus pleuropneumoniae APPΔclpPΔapxⅡCΔfur, they were challenged with virulent type 7 and type 5 strains. The results showed that APPΔclpPΔapxⅡCΔfur can provide good protection for mice and pigs.

The main advantage of the present invention is:

1. The material used in the present invention is Actinobacillus pleuropneumoniae serotype 7 CVCC265 strain, which is purchased from the China Veterinary Drug Control Institute. APP serotype 7 strains are currently the dominant serotype that is popular in my country and seriously causes pig disease. Therefore, in the future, vaccines developed based on the clpP, apxⅡC and fur three gene deletion mutant strains constructed by this strain will be highly targeted and have broad market application prospects.

2. The Actinobacillus pleuropneumoniae serotype 7 clpP, apxⅡC and fur gene deletion mutant strain of the present invention does not contain any resistance markers, and fully meets the vaccine biosafety requirements of my country.

Description of drawings

Fig. 1 is the construction flowchart of the recombinant suicide plasmid pUCΔclpP of Actinobacillus pleuropneumoniae;

Fig. 2 is a flowchart of the construction of the recombinant suicide plasmid pUCΔapxⅡC of Actinobacillus pleuropneumoniae;

Figure 3 is a flow chart of the construction of the recombinant suicide plasmid pUCΔfur of Actinobacillus pleuropneumoniae;

Fig. 4 is the PCR amplification result of upper homology arm clpPS and lower homology arm clpPX;

In the figure 1: clpPS (1200bp); 2: clpPX (1249bp); M: DL2000DNA Marker

Figure 5 is the PCR splicing result of the ΔclpP gene;

Figure 1: ΔclpP gene (2449bp); M: DL15000DNA Marker

Figure 6 is the PCR identification result of the recombinant suicide plasmid pUCΔclpP;

Figure 1-3: ΔclpP; M: DL15000 DNA Marker

Figure 7 is the PCR identification result of APPΔclpP single exchange strain;

Figure 1-3: Single exchange strain; M: DL2000DNA Marker

Figure 8 is the PCR identification result of the APPΔclpP deletion strain;

In the figure, M: DL2000 DNA Marker; No. 8 is the screened clpP single gene deletion strain

Figure 9 shows the results of the genetic stability experiment of the APPΔclpP deletion strain;

Figure 1-10: 1st-10th generation deletion strain; M: DL2000DNA Marker

Figure 10 is the PCR amplification result of the upper homology arm apxⅡCS and the lower homology arm apxⅡCX;

In the figure 1: apxⅡCS (1412bp); 2: apxⅡCX (1443bp); M: DL5000DNA Marker

Figure 11 is the PCR splicing result of ΔapxⅡC gene;

Figure 1: ΔapxⅡC gene (2855bp); M: DL5000DNA Marker

Figure 12 is the PCR identification result of the recombinant suicide plasmid pUCΔapxⅡC;

Figure 1-5: ΔapxⅡC; M: DL5000 DNA Marker

Figure 13 is the PCR identification result of APPΔclpPΔapxⅡC single exchange strain;

Figure 1-5: Single exchange strain; M: DL2000DNA Marker

Figure 14 is the PCR identification result of APPΔclpPΔapxⅡC double gene deletion strain;

In the figure, M: DL2000 DNA Marker; No. 1 and No. 5 are screened clpP and apxⅡC double gene deletion strains

Figure 15 is the result of the genetic stability experiment of the APPΔclpPΔapxⅡC double gene deletion strain;

Figure 1-10: 1st-10th generation deletion strain; M: DL2000DNA Marker

Figure 16 is the PCR amplification result of the upper homology arm furS and the lower homology arm furX;

Figure 1: furS (1387bp); 2: furX (1405bp); M: DL2000DNA Marker

Figure 17 is the PCR splicing result of Δfur gene;

Figure 1: Δfur gene (2792bp); M: DL5000DNA Marker

Figure 18 is the PCR identification result of the recombinant suicide plasmid pUCΔfur;

Figure 1-2: Δfur; M: DL5000 DNA Marker

Figure 19 is the PCR identification result of APPΔclpPΔapxⅡCΔfur single exchange strain;

Figure 1-4: Single exchange strain; M: DL2000DNA Marker

Figure 20 is the PCR identification result of APPΔclpPΔapxⅡCΔfur three-gene deletion strain;

In the figure, M: DL2000 DNA Marker; No. 5 is the screened clpP, apxⅡC and fur gene deletion strain

Figure 21 is the result of the genetic stability experiment of APPΔclpPΔapxⅡCΔfur three-gene deletion strain;

Figure 1-10: 1st-10th generation deletion strain; M: DL2000DNA Marker

Figure 22 shows the lung lesions of APPΔclpPΔapxⅡCΔfur three-gene deletion strain and parental strain after challenge.

Detailed ways

The present invention will be further described below in conjunction with specific examples, and the advantages and characteristics of the present invention will become clearer along with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Materials involved in the embodiments of the present invention:

Strain: APP serotype 7 strain CVCC265 purchased from China Veterinary Drug Control Institute

Vectors and reagents: pUC18 plasmid, Ex Taq DNA polymerase, and T4 DNA ligase were purchased from TaKaRa Company.

Example 1 Construction of APP Serum Type 7 clpP Single Gene Deletion Mutant

1.1 Construction of recombinant suicide vector pUCΔclpP

The flowchart for the construction of the recombinant suicide plasmid pUCΔclpP of Actinobacillus pleuropneumoniae is shown in Figure 1 .

1.1.1 Primer design and PCR amplification of the upper and lower homology arms of the ClpP protease gene

According to the reported sequence of APP7 AP76 strain (refer to the gene sequence of GenBank accession number CP001091.1), two pairs of primers were designed to amplify the upper homology arm clpPS and the lower homology arm clpPX of the clpP gene respectively, and the sizes of the amplified fragments were respectively 1200bp and 1249bp, the PCR amplification results of the upper homology arm clpPS and the lower homology arm clpPX are shown in FIG. 4 . An EcoRI restriction site was designed at the 5' end of the upstream primer of the upper homology arm, and a BamHI restriction site was designed at the 5' end of the downstream primer of the lower homology arm. The above primers were synthesized by Beijing Huada Gene Company.

The primer sequences for amplifying the upper homology arms are as follows:

clpSF:5'-CGGAATTC(EcoRI)GGGGCGTTACTGGATGC-3'

clpSR:5'- CCATCGCTTC CGCCTTTGGAGGTTTGC-3'

The primer sequences for the amplified lower homology arms are as follows:

clpXF:5'- TCCAAAGGCG GAAGCGATGGAATACGGTC-3'

clpXR:5'-CGGGATCC(BamHI)TTCTCTGCTTTAAGTGTCGGC-3'

The upper and lower homology arms of the clpP gene were respectively amplified using the APP serum type 7 CVCC265 strain genomic DNA as a template: the PCR amplification reaction system was 50 μL, including 10 ng of template DNA, 1 μM of upstream and downstream primers, 200 μM of dNTP, 10 μL of 10×Taq buffer, Ex Taq DNA Polymerase 2U (TaKaRa). The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 60s, 55°C for 60s, 72°C for 90s, and 72°C for 10 minutes. After gel electrophoresis, the PCR products were recovered with a gel extraction kit.

1.1.2 Overlap extension PCR method to amplify the ΔclpP gene fragment

The PCR recovery products of the upper and lower homology arms are used as templates, and the upper and lower homology arms of the clpP gene are spliced by the overlap extension PCR method, and the ΔclpP gene fragment is amplified. Part of the bases are reverse complementary, and the bases in the underlined part at the 5' end of primer clpXF are reverse complementary to the bases in the italic bold part of primer clpSR. The PCR amplification reaction system was 50 μL, including 10 ng of template DNA, 1 μM of upstream and downstream primers, 200 μM of dNTP, 10 μL of 10×Taq buffer, and 2U of Ex Taq DNA polymerase (TaKaRa). The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 60s, 55°C for 90s, 72°C for 150s, and 72°C for 10 minutes. After gel electrophoresis, the PCR product was recovered with a gel recovery kit, then digested with EcoRI and BamHI, and recovered with a gel recovery kit. The PCR splicing results of the ΔclpP gene are shown in Figure 5.

1.1.3 Construction of recombinant suicide plasmid pUCΔclpP

The purified ΔclpP gene fragment was ligated with T4 DNA ligase (TaKaRa) to the pUC18 vector digested by EcoRI and BamHI double enzymes, ligated at 16°C for 24 hours, heat-shocked and transformed into E. coli DH5α competent cells, which were identified as positive by colony PCR The clone was enriched and a small amount of plasmid was extracted by alkaline lysis method. The positive plasmid identified by enzyme digestion was named pUCΔclpP, and the cloned ΔclpP gene was identified by PCR and sequencing. The PCR identification result of the recombinant suicide plasmid pUCΔclpP is shown in Figure 6 .

1.2 Construction of APP serotype 7 clpP gene deletion mutant strain

The constructed recombinant suicide plasmid pUCΔclpP was electrotransformed into APP serum type 7 CVCC265, and the positive colonies of the single exchange strain were screened on a 10ug/mL Amp resistance plate, and primers were designed inside the upper and lower homology arms of clpP for PCR identification. The 858bp fragment can be amplified by the strain, the 367bp fragment can be amplified by the deletion strain, and the 858bp and 367bp fragment can be amplified by the single exchange strain at the same time. The PCR identification results of the APPΔclpP single exchange strain are shown in Figure 7. The above primers were synthesized by Beijing Huada Gene Company.

The primer sequences are as follows:

clpJDF: 5'-CGTGGTGTCGCTTGAAACTC-3'

clpJDR: 5'-AATTAGACCGTATTCCATCGC-3'

After the positive single colony of the single exchange strain was cultured and multiplied in TSB (1% nicotinamide adenine dinucleotide and 10% horse serum) without Amp resistance, it was spread on a non-resistant TSA plate, and picked Take a single colony and carry out PCR identification. The wild strain can amplify a fragment of 858bp, and the deletion strain can only amplify a fragment of 367bp. The PCR identification result of the APPΔclpP deletion strain is shown in Figure 8. The positive clone is named APPΔclpP.

The clpP gene deletion mutant strain APPΔclpP prepared by the present invention was continuously passaged on TSB medium for 10 times, and identified by PCR. If the mutant strain of each generation can amplify a 367bp fragment, it shows that the mutant strain of the present invention can be stably inherited; If the 858bp fragment is amplified, it shows that the mutant strain of the present invention cannot be inherited stably. The results are shown in Figure 9, indicating that the clpP gene deletion mutant strain prepared by the present invention can be stably inherited.

Example 2 Construction of APP serotype 7 clpP and apxⅡC double gene deletion mutants

2.1 Construction of recombinant suicide vector pUCΔapxⅡC

The flowchart for the construction of the recombinant suicide plasmid pUCΔapxⅡC of Actinobacillus pleuropneumoniae is shown in Figure 2.

2.1.1 Primer design and PCR amplification of the upper and lower homology arms of the apxⅡC gene

According to the reported sequence of APP7 AP76 strain (refer to the gene sequence of GenBank accession number CP001091.1), two pairs of primers were designed to amplify the upper homology arm apxⅡCS and the lower homology arm apxⅡCX of the apxⅡC gene respectively, and the sizes of the amplified fragments were respectively 1412bp and 1443bp, the PCR amplification results of the upper homology arm apxⅡCS and the lower homology arm apxⅡCX are shown in FIG. 10 . An EcoRI restriction site was designed at the 5' end of the upstream primer of the upper homology arm, and a BamHI restriction site was designed at the 5' end of the downstream primer of the lower homology arm. The above primers were synthesized by Beijing Huada Gene Company.

The primer sequences for amplifying the upper homology arms are as follows:

Ⅱ CSF: 5'-CGGAATTC(EcoR)ATGACAACACCAATGATTGATTTAC-3'

Ⅱ CSR: 5'- AATCCCCGAA AGCATCATCCCTCCCATTC-3'

The primer sequences for the amplified lower homology arms are as follows:

Ⅱ CXF: 5'- GGATGATGCT TTCGGGGATTCATCTCTATTG-3'

Ⅱ CXR: 5'-CGGGATCC(BamH)GTTGTAATAAGTCCCGTAACACCAG-3'

The upper and lower homology arms of the apxⅡC gene were respectively amplified using the genomic DNA of APP serum type 7 strain CVCC265 as a template: the PCR amplification reaction system was 50 μL, including 10 ng of template DNA, 1 μM of upstream and downstream primers, 200 μM of dNTP, 10 μL of 10×Taq buffer, Ex Taq DNA Polymerase 2U (TaKaRa). The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 60s, 55°C for 60s, 72°C for 90s, and 72°C for 10 minutes. After gel electrophoresis, the PCR products were recovered with a gel extraction kit.

2.1.2 Overlap extension PCR method to amplify the ΔapxⅡC gene fragment

The PCR recovery products of the upper and lower homology arms were used as templates, and the upper and lower homology arms of the apxⅡC gene were spliced by the overlap extension PCR method to amplify the ΔapxⅡC gene fragment. The PCR splicing results of the ΔapxⅡC gene are shown in Figure 11. The underlined base at the 5' end of primer II CSR is reverse complementary to the base in italic bold of primer II CXF, and the base underlined at the 5' end of primer II CXF is reverse complementary to the base in italic bold of primer IICSR. The PCR amplification reaction system was 50 μL, including 10 ng of template DNA, 1 μM of upstream and downstream primers, 200 μM of dNTP, 10 μL of 10×Taq buffer, and 2U of Ex Taq DNA polymerase (TaKaRa). The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 60s, 55°C for 90s, 72°C for 150s, and 72°C for 10 minutes. After gel electrophoresis, the PCR product was recovered with a gel recovery kit, then digested with EcoRI and BamHI, and recovered with a gel recovery kit.

2.1.3 Construction of recombinant suicide plasmid pUCΔapxⅡC

The purified ΔapxⅡC gene fragment was ligated with T4 DNA ligase (TaKaRa) to the pUC18 vector digested by EcoRI and BamHI double enzymes, ligated at 16°C for 24 hours, heat-shocked and transformed into Escherichia coli DH5α competent cells, which were identified as positive by colony PCR The clone was enriched and a small amount of plasmid was extracted by alkaline lysis method. The positive plasmid identified by enzyme digestion was named pUCΔapxⅡC, and the cloned ΔapxⅡC gene was identified by PCR and sequencing. The PCR identification result of the recombinant suicide plasmid pUCΔapxⅡC is shown in Figure 12 .

2.2 Construction of APP serotype 7 clpP and apxⅡC double gene deletion mutants

Electrotransform the constructed recombinant suicide plasmid pUCΔapxⅡC into the APPΔclpP constructed in Example 1, screen the positive colonies of the single exchange strain on a 10ug/mL Amp resistance plate, and design primers inside the upper and lower homology arms of apxⅡC, and perform PCR Identification, the clpP single gene deletion strain can amplify a 564bp fragment, the clpP and apxⅡC double gene deletion strain can amplify a 294bp fragment, the single exchange strain can simultaneously amplify a 564bp and 294bp fragment, the PCR identification results of the APPΔclpPΔapxⅡC single exchange strain are as follows Figure 13 shows. The above primers were synthesized by Beijing Huada Gene Company.

The primer sequences are as follows:

IICJDF: 5'-GAAGAGCCATTACCCAACAAC-3'

IICJDR: 5'-ATACAATAGAGATGAATCCCCG-3'

After the positive single colony of the single exchange strain was cultured and multiplied in TSB (1% nicotinamide adenine dinucleotide and 10% horse serum) without Amp resistance, it was spread on a non-resistant TSA plate, and picked Take a single colony and perform PCR identification. The clpP single gene deletion strain can amplify a 564bp fragment, and the clpP and apxⅡC double gene deletion strain can only amplify a 294bp fragment. The PCR identification results of the APPΔclpPΔapxⅡC double gene deletion strain are shown in Figure 14. The positive clone was named APPΔclpPΔapxⅡC.

The clpP and apxⅡC double-gene deletion mutant strain APPΔclpPΔapxⅡC prepared by the present invention is continuously passaged on TSB medium for 10 times, and identified by PCR. If the mutant strain of each generation can amplify a 294bp fragment, it shows that the mutant strain of the present invention is capable of stabilizing Inherited; if a 564bp fragment is amplified, it indicates that the mutant strain of the present invention cannot be inherited stably. The results are shown in Figure 15, indicating that the clpP and apxIIC double gene deletion mutant strain prepared by the present invention can be stably inherited.

Example 3 Construction of APP serotype 7 gene deletion mutants of clpP, apxⅡC and fur

3.1 Construction of recombinant suicide vector pUCΔfur

The flowchart for the construction of the recombinant suicide plasmid pUCΔfur of Actinobacillus pleuropneumoniae is shown in Figure 3 .

3.1.1 Primer design and PCR amplification of upper and lower homology arms of fur gene

According to the reported sequence of APP7 AP76 strain (refer to the gene sequence of GenBank accession number CP001091.1), two pairs of primers were designed to amplify the upper homology arm furS and the lower homology arm furX of the fur gene respectively, and the sizes of the amplified fragments were respectively 1412bp and 1443bp, the PCR amplification results of the upper homology arm furS and the lower homology arm furX are shown in FIG. 16 . An EcoRI restriction site was designed at the 5' end of the upstream primer of the upper homology arm, and a BamHI restriction site was designed at the 5' end of the downstream primer of the lower homology arm. The above primers were synthesized by Beijing Huada Gene Company.

The primer sequences for amplifying the upper homology arms are as follows:

furSF:5'-CGGAATTC(EcoR)TTAGCCGTGATGGTGGTG-3'

furSR: 5'- TTTCAT GCATTTTCTTCAGACATAACTTG-3'

The primer sequences for the amplified lower homology arms are as follows:

furXF:5'- AAGAAAATGC ATGAAATTAGCGACGCACAG-3'

furXR:5'-CGGGATCC(BamH)GTTTAGGCTCGCCTTTGC-3'

The upper and lower homology arms of the fur gene were respectively amplified using the genomic DNA of APP serum type 7 CVCC265 strain as a template: the PCR amplification reaction system was 50 μL, including 10 ng of template DNA, 1 μM of upstream and downstream primers, 200 μM of dNTP, 10 μL of 10×Taq buffer, Ex Taq DNA Polymerase 2U (TaKaRa). The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 60s, 55°C for 60s, 72°C for 90s, and 72°C for 10 minutes. After gel electrophoresis, the PCR products were recovered with a gel extraction kit.

3.1.2 Overlap extension PCR method to amplify the Δfur gene fragment

The PCR recovery products of the upper and lower homology arms were used as templates, and the upper and lower homology arms of the fur gene were spliced by the overlap extension PCR method to amplify the Δfur gene fragment. The PCR splicing result of the Δfur gene is shown in FIG. 17 . The underlined base at the 5' end of the primer furSR is reverse complementary to the base in the italic bold part of the primer furXF, and the base at the underlined part at the 5' end of the primer furXF is reverse complementary to the base in the italic bold part of the primer fur SR. The PCR amplification reaction system was 50 μL, including 10 ng of template DNA, 1 μM of upstream and downstream primers, 200 μM of dNTP, 10 μL of 10×Taq buffer, and 2U of Ex Taq DNA polymerase (TaKaRa). The PCR reaction program was: 95°C pre-denaturation for 5 minutes, 30 cycles at 94°C for 60s, 55°C for 90s, 72°C for 150s, and 72°C for 10 minutes. After gel electrophoresis, the PCR product was recovered with a gel recovery kit, then digested with EcoRI and BamHI, and recovered with a gel recovery kit.

3.1.3 Construction of recombinant suicide plasmid pUCΔfur

The purified Δfur gene fragment was ligated with T4 DNA ligase (TaKaRa) to the pUC18 vector digested by EcoRI and BamHI double enzymes, ligated at 16°C for 24 hours, heat-shocked and transformed into E. coli DH5α competent cells, which were identified as positive by colony PCR The clone was enriched and a small amount of plasmid was extracted by alkaline lysis method. The positive plasmid identified by enzyme digestion was named pUCΔfur, and the cloned Δfur gene was identified by PCR and sequencing. The PCR identification result of the recombinant suicide plasmid pUCΔfur is shown in Figure 18 .

3.2 Construction of APP serotype 7 gene deletion mutants of clpP, apxⅡC and fur

The constructed recombinant suicide plasmid pUCΔfur was electrotransformed into the APPΔclpPΔapxIIC constructed in Example 2, and the positive colonies of the single exchange strain were screened on a 10ug/mL Amp resistance plate, and primers were designed inside the upper and lower homology arms of fur, and PCR was carried out Identification, clpP and apxⅡC double-gene deletion strains can amplify 897bp fragments, clpP, apxⅡC and fur three-gene deletion strains can amplify 548bp fragments, single exchange strains can simultaneously amplify 897bp and 548bp fragments, APPΔclpPΔapxⅡCΔfur three-gene deletion strains The PCR identification results are shown in Figure 19. The above primers were synthesized by Beijing Huada Gene Company.

The primer sequences are as follows:

furJDF: 5'-GACATTGGCCGACGGAAG-3'

furJDR: 5'-GCCAATCACGAAAGCAACG-3'

After the positive single colony of the single exchange strain was cultured and multiplied in TSB (1% nicotinamide adenine dinucleotide and 10% horse serum) without Amp resistance, it was spread on a non-resistant TSA plate, and picked Take a single colony and carry out PCR identification. The clpP and apxⅡC double-gene deletion strain can amplify and obtain a 897bp fragment, and the clpP, apxⅡC and fur three-gene deletion strain can only amplify a 548bp fragment. The PCR identification results of the APPΔclpPΔapxⅡCΔfur three-gene deletion strain are shown in the figure As shown in 20, the positive clone was named APPΔclpPΔapxⅡCΔfur, and was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms, and its preservation number was CGMCC No.7930.

The clpP, apxⅡC and fur three-gene mutant strain APPΔclpPΔapxⅡCΔfur prepared by the present invention were continuously passaged on TSB medium for 10 times, and identified by PCR. If the mutant strain of each generation can amplify a 548bp fragment, it shows that the mutant strain of the present invention is able to Stable inheritance; if a 897bp fragment is amplified, it indicates that the mutant strain of the present invention cannot be stably inherited. The results are shown in Figure 21, indicating that the clpP, apxIIC and fur three gene deletion mutant strains prepared by the present invention can be stably inherited.

Example 4 Toxicity identification and safety evaluation of APPΔclpPΔapxⅡCΔfur mutant strain

Experimental animals: SPF grade Balb/C female mice aged 4-6 weeks, APP seronegative healthy pigs aged 8-9 weeks were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

4.1 Toxicity identification and safety evaluation test on mice

The mice were randomly divided into two groups, 60 in each group. The specific vaccination plan is as follows:

The first group (test group): inoculated with APPΔclpPΔapxⅡCΔfur prepared in Example 3, diluted to 6 concentrations (CFU) listed in Table 1, 10 mice for each concentration, and each mouse was inoculated intraperitoneally with a dose of 0.1ml.

The second group (control group): inoculated with Actinobacillus pleuropneumoniae CVCC265, diluted to 6 concentrations (CFU) listed in Table 1, each concentration of 10 mice, each mouse intraperitoneal inoculation dose 0.1ml.

The survival of the mice is shown in Table 1. The results showed that the toxicity of APPΔclpPΔapxⅡCΔfur to mice was more than 100 times lower than that of the wild strain CVCC265.

Table 1

4.2 Toxicity identification and safety evaluation test on pigs

The pigs were randomly divided into two groups with 9 pigs in each group. The specific vaccination plan is as follows:

The first group (test group): inoculated with the APPΔclpPΔapxⅡCΔfur prepared in Example 3, diluted to 3 concentrations (CFU) listed in Table 2, 3 pigs for each concentration, and 1 ml of intratracheal inoculation dose for each pig.

The second group (control group): inoculated with Actinobacillus pleuropneumoniae CVCC265, diluted to 3 concentrations (CFU) listed in Table 2, each concentration of 3 pigs, each pig intratracheal inoculation dose 1ml.

The body temperature was monitored within one week after the challenge, and the body temperature of the pigs inoculated with the APPΔclpPΔapxⅡCΔfur prepared by the present invention was normal, between 38.7-39.6°C, not exceeding 40°C; while the body temperature of the pigs in the control group generally rose, and some pigs had a body temperature as high as 41.5°C. Spend. During this period, the mental appetite of the pigs in the test group was normal, and no abnormal changes were seen; while the pigs in the control group had poor mental appetite.

See Table 2 for the survival and disease status of the pigs. The results showed that the toxicity of APP△clpP△apxⅡC△fur to pigs was more than 100 times lower than that of the wild strain CVCC265.

Table 2

Two weeks after the challenge, the pigs were anesthetized and killed, and the lung lesions were observed. The results showed that the lungs of the test group were relatively normal without obvious lesions; while severe bleeding was seen in the control group, forming large bleeding spots or purulent foci, as shown in Figure 22.

Example 5 Immune protection test of APPΔclpPΔapxⅡCΔfur mutant strain

Experimental animals: 4-6 weeks old SPF grade Balb/C female mice, 4-5 weeks old APP seronegative weaned piglets, all purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

5.1 Mice immune protection test

The mice were randomly divided into three groups, 10 in each group. The specific vaccination plan is as follows:

The first group (test group 1): inoculated with APPΔclpPΔapxⅡCΔfur prepared in Example 3, each mouse was inoculated intraperitoneally with a dose of 0.1 ml, containing 1×10 ⁶ CFU of bacteria.

The second group (experimental group 2): inoculate inactivated Actinobacillus pleuropneumoniae CVCC265 (emulsified as inactivated vaccine with Freund's adjuvant), every mouse intraperitoneal inoculation dose 0.1ml, count its inside before inactivation Contains 2×10 ⁹ CFU bacteria.

The third group (control group): inoculate TSB liquid culture medium, every mouse intraperitoneally inoculates dose 0.1ml.

Four weeks after immunization, mice in each group were challenged with APP serotype 7 CVCC265 bacteria (live bacteria content 1×10 ⁷ CFU). Table 3 shows the survival of the mice.

table 3

test group Survival number of mice protection rate APPΔclpPΔapxⅡCΔfur 10/10 100% CVCC265 inactivated vaccine 5/10 50% TSB 0/10 0

5.2 Pig immune protection test

The pigs were randomly divided into three groups, 10 in each group. The specific vaccination plan is as follows:

The first group (three gene deletion strain attenuated live vaccine group): intramuscular injection of APPΔclpPΔapxⅡCΔfur prepared in Example 3, dose 1ml (live bacteria content 2×10 ⁷ CFU), 21 days after immunization, booster immunization once with the same dose.

The second group (inactivated vaccine group): intramuscular injection of 0.3% formaldehyde inactivated CVCC265 (inactivated vaccine emulsified with complete Freund's adjuvant), the dose is 1ml (dead bacteria content 2×10 ¹⁰ CFU), 21 days after immunization Every day, booster immunization once with the same dose (the adjuvant for the second immunization is Freund's incomplete adjuvant).

The third group (control group): no vaccination, normal feeding.

21 days after the second immunization, the trachea of APP7-type CVCC265 and 5-type MD12 strains were challenged, with 5 pigs in each group, and the doses were 5×10 ⁹ CFU and 1×10 ⁹ CFU, respectively. Each pig was injected with 1ml, and observed after the challenge. week. See Table 4 for pig survival and morbidity.

Table 4

Claims (6)

1. Application of a three-gene deletion strain of Actinobacillus pleuropneumoniae without a resistance marker in the preparation of a vaccine for preventing porcine infectious pleuropneumonia, characterized in that the bacterial strain lacks protease ClpP, ApxⅡtoxin activator ApxⅡC and iron The gene encoding the regulatory protein Fur is absorbed, and the Actinobacillus pleuropneumoniae is a serotype 7 strain of Actinobacillus pleuropneumoniae. 2. The application according to claim 1, characterized in that said Actinobacillus pleuropneumoniae is Actinobacillus pleuropneumoniae serotype 7 strain CVCC265. 3. The application according to claim 1, characterized in that: the amino acid sequence of the protease ClpP is as shown in SEQ ID NO.2, and the amino acid sequence of the ApxII toxin activator ApxIIC is as shown in SEQ ID NO.4, The amino acid sequence of the iron uptake regulatory protein Fur is shown in SEQ ID NO.6. 4. The application according to any one of claims 1-3, characterized in that: the gene deletion strain is APP-clpPΔapxⅡCΔfur, which is preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms, and its preservation number is CGMCC No .7930. 5. A three-gene deletion strain of Actinobacillus pleuropneumoniae without a resistance marker, characterized in that: the gene deletion strain is APPΔclpPΔapxⅡCΔfur, which is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and its preservation number is It is CGMCC No.7930. 6. A porcine infectious pleuropneumonia attenuated live vaccine without a resistance marker, whose active ingredient is the three-gene deletion strain of Actinobacillus pleuropneumoniae without a resistance marker according to claim 5.