Construction of porcine actinobacillus pleuropneumoniae serum type 1 attenuated mutant strain and application of porcine actinobacillus pleuropneumoniae serum type 1 attenuated mutant strain in vaccine preparation

Abstract

The invention discloses construction of a porcine actinobacillus pleuropneumoniae serum type 1 attenuated mutant strain and application of the porcine actinobacillus pleuropneumoniae serum type 1 attenuated mutant strain in vaccine preparation, and relates to the technical field of gene deletion strain research. Comprising the following steps: researching a poison preparation factor Apx toxin of each serotype in actinobacillus pleuropneumoniae through literature search, amplifying upstream and downstream homologous arms of an Apx toxin gene through fusion PCR (Polymerase Chain Reaction), constructing a suicide plasmid, and screening through conjugational transfer to obtain a serum type 1 attenuated mutant strain. The attenuated engineering strain simultaneously expresses the three toxins, the toxicity is obviously reduced, the cross protection range is expanded, and the key is that the immune effect of the mutant strain is superior to that of an MSD vaccine.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering in biotechnology, and specifically relates to the construction of an attenuated mutant strain of Actinobacillus pleuropneumoniae serotype 1 and its application in vaccine preparation. Background Technology

[0002] Porcine contagious pleuropneumonia (APP), caused by Actinobacillus pleuropneumoniae, is a persistent respiratory disease in pigs, causing acute, acute, and chronic infections and resulting in significant economic losses to the pig industry globally. Nineteen serotypes have been identified, with different prevalent serotypes in different regions. In China, serotypes 1, 3, and 7 are more common, while the prevalence of serotypes 5, 10, 11, and 15 is gradually increasing. Prevention of APP mainly involves the use of drugs and vaccines. Developing vaccines with good immunization efficacy and cross-protection is of great significance. Currently available commercial vaccines include inactivated vaccines, live attenuated vaccines, and subunit vaccines. The bivalent inactivated vaccine for porcine streptococcal disease and contagious pleuropneumonia (serotype 2 ZY-2 strain + serotype 1 SC strain) developed by Huapai Biotechnology Group Co., Ltd. primarily targets the prevalent serotypes of Streptococcus suis type 2 and APP type 1 in my country. Wuhan Keqian Biotechnology Co., Ltd. has developed a trivalent inactivated vaccine (types 1, 2, and 7) and a gene-deleted vaccine (HB-04M strain) for porcine contagious pleuropneumonia (APP) to prevent infection caused by APP types 1, 2, and 7. These vaccines were imported from Merck Animal Health. The Aipuke vaccine is the first APP subunit toxoid vaccine in China. It contains one outer membrane protein (OMP) secreted by APP and three toxoid antigens (ApxI, ApxII, and ApxIII), and can prevent infection of 15 serotypes.

[0003] APP has multiple serotypes, and there is a lack of cross-protection between them. Domestically produced vaccines are low-cost, easy to promote and apply, but they can only provide protection against some serotypes, and their protection against the newly emerging serotypes 5 and 15 may be limited. Imported subunit vaccines can simultaneously express all three APP toxins (ApxI, ApxII, and ApxIII), can prevent multiple serotypes, and have good immunization effects, but they are expensive and not suitable for large-scale use in pig farms. Apx toxins are the key virulence factors of APP, including four main types: ApxI, ApxII, ApxIII, and ApxIV. ApxIV has a unique expression pattern—it is present in all serotypes, but is only expressed in the infected host, and not under in vitro culture conditions. This characteristic makes it an ideal diagnostic marker for distinguishing between naturally infected and vaccine-immunized animals. Each serotype can only express one or two of the ApxI-III toxins. For example, highly pathogenic serotypes 1 and 5 express ApxI and ApxII, while serotypes 3 and 15 express ApxII and ApxIII. This difference in expression pattern directly affects the pathogenicity of different strains. The Apx toxin operon includes four genes: CA, B, and C. The A gene encodes the structural protein of the toxin; the synthesized A protein does not possess toxic activity. The C gene typically binds to the A gene to form the correct conformation, activating its toxic activity. Genes B and D are responsible for toxin transport and secretion. ApxI and ApxIII have all four genes (CA, B, and C), while ApxII only has the CA gene and lacks autonomous secretion ability, relying on the B and D genes of ApxI or III for transmembrane transport. Summary of the Invention

[0004] One objective of this invention is to provide an attenuated mutant strain of Actinobacillus pleuropneumoniae, WH01-3A (APPΔapxIVΔapxICΔapxIIC / apxIIIA). + This invention utilizes a clinical type 1 strain as the parent strain, deleting the apxIC, apxIIC, and apxIV genes. This allows the expressed ApxI and ApxII to retain immunogenicity while losing toxicity, resulting in a significant decrease in virulence. Furthermore, by inserting an exogenous apxIIIA gene, an attenuated engineered strain capable of simultaneously expressing all three toxins is constructed. This strategy significantly reduces the strain's virulence while expanding its cross-protective range.

[0005] The second objective of this invention is to provide an attenuated mutant strain of Actinobacillus pleuropneumoniae, WH01-3A (APPΔapxIVΔapxICΔapxIIC / apxIIIA). +The construction method of ) was as follows: Using the porcine Actinobacillus pleuropneumoniae serotype 1 strain WH01 as the maternal strain, the apxIV, apxIC, and apxIIC genes in the genome were sequentially deleted through homologous recombination. Then, the promoter of the nap gene and the apxIIIA gene were inserted at the position of the deleted apxIV gene to obtain the attenuated mutant strain WH01-3A.

[0006] The third objective of this invention is to provide a live attenuated vaccine for porcine infectious pleuropneumonia, the active ingredient of which is the attenuated mutant strain WH01-3A of Actinobacillus pleuropneumoniae described in this invention.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] 1. First, using the clinical strain WH01 of Actinobacillus pleuropneumoniae serotype 1 as the main construction material, the apxIV-up fragment, apxIV-down fragment, apxIC-up fragment, apxIC-down fragment, apxIIC-up fragment, apxIIC-down fragment, and the promoter (Pnap) fragment of the nap gene were amplified from the genome of strain WH01 by PCR. The apxIIIA gene fragment was amplified from the genome of strain JL03. The apxIV-up and apxIV-down fragments were ligated into the pEMOC2 vector to obtain the recombinant plasmid pEMOC2-ΔapxIV; the apxIC-up and apxIC-down fragments were ligated into the pEMOC2 vector to obtain the recombinant plasmid pEMOC2-ΔapxIC; the apxIIC-up and apxIIC-down fragments were ligated into the pEMOC2 vector to obtain the recombinant plasmid pEMOC2-ΔapxIIC; and the apxIV-up, Pnap, apxIIIA, and apxIV-down fragments were ligated into the enzyme-digested pEMOC2 vector to obtain the recombinant plasmid pEMOC2-Pnap+apxIIIA. The constructed recombinant plasmids were then transformed into *E. coli* β2155 competent cells using chemical transformation, and positive clones were selected for identification.

[0009] 2. After co-culturing pEMOC2-ΔapxIV positive clones with WH01, single-exchange positive bacteria were identified. These single-exchange positive bacteria were then subjected to reverse selection using the sacB gene and PCR identification, yielding double-exchange positive bacteria, namely the APPΔapxIV strain. Further, after co-culturing pEMOC2-ΔapxIC positive clones with APPΔapxIV, single-exchange positive bacteria were identified. These single-exchange positive bacteria were then subjected to reverse selection using the sacB gene and PCR identification, yielding double-exchange positive bacteria, namely the APPΔapxIVΔapxIC strain. Further, after co-culturing pEMOC2-ΔapxIIC positive clones with APPΔapxIVΔapxIC strain, single-exchange positive bacteria were identified. These single-exchange positive bacteria were then subjected to reverse selection using the sacB gene and PCR identification, yielding double-exchange positive bacteria, namely the APPΔapxIVΔapxICΔapxIIC strain. Finally, after co-culturing pEMOC2-Pnap+apxIIIA positive clones with APPΔapxIVΔapxICΔapxIIC clones, single-exchange positive bacteria were identified. These single-exchange positive bacteria were then subjected to reverse selection using the sacB gene and PCR identification to obtain double-exchange positive bacteria, resulting in APPΔapxIVΔapxICΔapxIIC / apxIIIA. + The strain was identified and named WH01-3A.

[0010] The attenuated mutant strain WH01-3A provided by this invention has a virulence reduction of about 150 times compared with the parent strain WH01, while maintaining the same growth rate and significantly reducing hemolytic activity. Furthermore, this strain can be stably inherited.

[0011] Compared to existing technologies, the advantages of this invention are:

[0012] 1. The maternal strain used in this invention is a clinically isolated serotype 1 strain of Actinobacillus pleuropneumoniae, named WH01. Serotype 1 is one of the main prevalent serotypes of APP in my country. The mutant strain obtained after genetic engineering modification has significantly reduced virulence.

[0013] 2. The WH01-3A strain constructed in this invention can simultaneously express toxins apxIA, apxIIA, and apxIIIA. Currently, none of the serotypes of APP can simultaneously express all three toxins. Apx is an important virulence factor for APP and also an important candidate antigen for APP vaccines.

[0014] 3. The WH01-3A strain of the present invention does not contain any resistance markers and fully complies with my country's vaccine biosafety requirements.

[0015] 4. The WH01-3A strain provided by this invention has approximately 150 times less virulence than the maternal strain WH01, but its growth rate is unaffected, while its hemolytic activity is significantly reduced. Mice immunized with the WH01-3A strain all produced antibodies against ApxIA, ApxIIA, and ApxIIIA. After challenge with the maternal strain WH01, clinical serum type 5, and type 15 strains, the survival rates of mice in the WH01-3A immunization group were 87.5%, 75%, and 87.5%, respectively, while the survival rates of mice in the imported subunit vaccine (MSD immunization group) were 50%, 75%, and 87.5%, respectively. The attenuated WH01-3A strain showed better immunization efficacy than the MSD vaccine. Attached Figure Description

[0016] Figure 1 The results of PCR identification of positive clones of recombinant plasmids are shown. M is the 5000 DNA Marker. In Figure A, 1: pEMOC2 empty vector, 2-4: pEMOC2-ΔapxIV, 5-7: pEMOC2-ΔapxIC, 8-9: pEMOC2-ΔapxIIC; in Figure B, 1-3: pEMOC2-Pnap+apxIIIA; 4: pEMOC2 empty vector.

[0017] Figure 2 A schematic diagram illustrating the construction of a gene-deleted strain.

[0018] Figure 3 A schematic diagram of the construction of strain APPΔapxIVΔapxICΔapxIIC by inserting the Pnap+apxIIIA gene.

[0019] Figure 4 The results are PCR identification results from the construction of the attenuated mutant strain. M represents the 5000 DNA Marker.

[0020] A-1 is the identification result of single-exchange primers apxIV-UF / apxIV-DR.

[0021] A-2 shows the results of double exchange identification. In the figure, 1-3: identification of primers apxIV-inner F / apxIV-inner R, where 1 is APPΔapxIV, 2 is WH01, and 3 is the negative control; 4-6: identification of primers apxIV-UF / apxIV-DR, where 4 is APPΔapxIV, 5 is WH01, and 6 is the negative control.

[0022] B-1 is the result of single-exchange primer identification for apxIC-UF / apxIC-DR.

[0023] B-2 shows the results of double exchange identification. In the figure, 1-3: identification of primers apxIC-inner F / apxIC-inner R, where 1: APPΔapxIVΔapxIC, 2: WH01, 3: negative control; 4-6: identification of primers apxIC-UF / apxIC-DR, where 4: APPΔapxIVΔapxIC, 5: WH01, 6: negative control.

[0024] C-1 is the identification result of single-exchange primer apxIIC-UF / apxIIC-DR.

[0025] C-2 represents identification of primers apxIIC-inner F / apxIIC-inner R in the figure, where 1: APPΔapxIVΔapxICΔapxIIC, 2: WH01, and 3: negative control; 4-6 represent identification of primers apxIIC-UF / apxIIC-DR, where 4: APPΔapxIVΔapxICΔapxIIC, 5: WH01, and 6: negative control.

[0026] D-1 is the identification result of single exchange primer apxIV-UF / apxIV-DR.

[0027] D-2 shows the results of double exchange identification. In the figure, 1-3: identification of primers apxIV-UF / apxIV-DR, where 1: APPΔapxIVΔapxICΔapxIIC / apxIIIA+, 2: WH01, 3: negative control; 4-6: identification of primers Pnap-inner F / apxIIIA-inner R, where 4: APPΔapxIVΔapxICΔapxIIC / apxIIIA+, 5: WH01, 6: negative control.

[0028] In Figure E, 1-3: primers apxIV-inner F / apxIV-inner R for identification; 4-6: primers apxIV-UF / apxIV-DR for identification; 7-9: primers Pnap-inner F / apxIIIA-inner R for identification; 10-12: primers apxIC-inner F / apxIC-inner R for identification; 13-15: primers apxIC-UF / apxIC-DR for identification; 16-18: primers apxIIC-inner F / apxIIC-inner R for identification; and 19-21: primers apxIIC-UF / apxIIC-DR for identification. Among these, 1, 4, 7, 10, 13, 16, and 19 are WH01-3A; 2, 5, 8, 11, 14, 17, and 20 are WH01; and 3, 6, 9, 12, 15, 18, and 21 are negative controls.

[0029] Figure 5 The growth curves of the mutant strain WH01-3A and the parent strain WH01 are shown.

[0030] Figure 6 The results show the hemolytic activity of the mutant strain WH01-3A and the parent strain WH01.

[0031] Figure 7 Figure 1 shows the PCR results for identifying the genetic stability of mutant strain WH01-3A. M represents either a 2000 DNA Marker or a 15000 DNA Marker. Figure A shows the identification results using primers apxIC-innerF / apxIC-innerR, where 1-10 represent generations 2-20 of mutant strain WH01-3A, 11 represents WH01, and 12 represents the negative control. Figure B shows the identification results using primers apxIC-UF / apxIC-DR, where 1-10 represent generations 2-20 of mutant strain WH01-3A, 11 represents WH01, and 12 represents the negative control. Figure C shows the identification results using primers apxIIC-innerF / apxIIC-innerR, where 1-10 represent generations 2-20 of mutant strain WH01-3A, 11 represents WH01, and 12 represents the negative control. Figure D shows the identification results of apxIIC-UF / apxIIC-DR, where 1-10: mutant strain WH01-3A, generations 2 to 20; 11: WH01; 12: negative control. Figure E shows the identification results of primers apxIV-innerF / apxIV-innerR, where 1-10: mutant strain WH01-3A, generations 2 to 20; 11: WH01; 12: negative control. Figure F shows the identification results of primers Pnap-innerF / apxIIIA-innerR, where 1-10: mutant strain WH01-3A, generations 2 to 20; 11: WH01; 12: negative control.

[0032] Figure 8 The results show the antibody levels in mice immunized with the mutant strain WH01-3A and the commercial vaccine. Figure A shows the antibody results against type 1 whole bacterial protein, Figure B shows the antibody results against type 5 whole bacterial protein, Figure C shows the antibody results against type 15 whole bacterial protein, Figure D shows the antibody results against protein ApxIA, Figure E shows the antibody results against protein ApxIIA, and Figure F shows the antibody results against protein ApxIIIA.

[0033] Figure 9 The immune protection rates of mice immunized with mutant strain WH01-3A and commercially available vaccines against different serotype strains are shown in Figure A, which shows the results of challenge with serotype 1 strain WH01; Figure B shows the results of challenge with serotype 5 strain; and Figure C shows the results of challenge with serotype 15 strain. Detailed Implementation

[0034] The invention will be explained below through specific examples.

[0035] Experimental materials and reagents:

[0036] 1. Strains and plasmids

[0037] The clinical serum strains of Actinobacillus pleuropneumoniae (APP) type 1 (WH01), type 5, and type 15 were isolated and preserved by the laboratory of Li Lu at Huazhong Agricultural University. The standard serum strain of Actinobacillus pleuropneumoniae (APP) type 3 (JL03) (GenBank accession number: CP000687.1) and the pEMOC2 conjugation transfer plasmid (GenBank accession number: AJ868288.1) were preserved by the laboratory of Li Lu at Huazhong Agricultural University.

[0038] 2. Culture medium and reagents

[0039] Tryptic soy agar (TSA) and tryptic soy broth (TSB) were purchased from BD Difco and prepared according to the instructions.

[0040] Nicotinamide adenine dinucleotide (NAD): Prepare a 10 mg / mL stock solution and store it at -20°C. Add 0.1% NAD to TSA or TSB by weight / volume percentage.

[0041] Fresh Mianyang blood: Prepare blood agar plates by adding 5% Mianyang blood to TSA by weight and volume percentage.

[0042] LB and LA media: Prepare according to the instructions.

[0043] Chloramphenicol (Cm): The concentrations used were 20 μg / mL and 2 μg / mL, respectively;

[0044] Diaminopimelic acid (DAP): Prepare a 1M stock solution and store it at -20℃. Add 0.1% DAP to LB or LA by mass-volume percentage.

[0045] Magnesium sulfate (MgSO4): Prepare a 1M stock solution and store it at -20℃. Add 0.1% MgSO4 to LA or LB by mass-volume percentage.

[0046] Sucrose: Prepare a plate for screening single exchange by adding 10% sucrose to TSA according to the mass-volume percentage. 3. Primers.

[0047] Table 1. All primers used in this experiment

[0048]

[0049]

[0050] 4. Laboratory animals

[0051] The 6-week-old Balb / c female mice and the 4-week-old KM female mice used in this experiment were purchased from the Experimental Animal Center of Huazhong Agricultural University.

[0052] Example 1: Construction of porcine Actinobacillus pleuropneumoniae serum type 1 strain WH01-3A

[0053] 1. Construction of recombinant suicide plasmid

[0054] (1) Fragment amplification and product recovery

[0055] Using the genome of strain WH01 as a template, the upstream fragment of apxIV (1067 bp) was amplified using primers upstream-F / R and the downstream fragment of apxIV (1075 bp) was amplified using primers downstream-F / R.

[0056] The upstream fragment of apxIC (1129 bp) was amplified using primers upstream-F / R and the downstream fragment of apxIC (1145 bp) was amplified using primers downstream-F / R.

[0057] The upstream fragment of apxIIC (1151 bp) was amplified using primers upstream-F / R and the downstream fragment of apxIIC (1114 bp) was amplified using primers downstream-F / R.

[0058] A 400bp Pnap fragment was amplified using primers Pnap fragment-F / R.

[0059] Using the genome of strain JL03 as a template, a 3159bp apxIIIA fragment was amplified from the genome of strain JL03 using apxIIIA fragment-F / R primers. The primer sequences are shown in Table 1.

[0060] Fragment amplification was performed using a 50 μL PCR reaction mixture: 25 μL 2×Super Kfx MasterMix, 1 μL upstream primer, 1 μL downstream primer, 1 μL genomic DNA, and 22 μL ddH2O. The PCR program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 20 s, 30 cycles, followed by a final 10 min at 72℃. The PCR products were recovered using a gel extraction kit after gel electrophoresis. The recovered products were stored at -20℃.

[0061] (2) Enzyme digestion and recovery of empty vector plasmids

[0062] The pEMOC2 empty vector plasmid was digested using restriction endonucleases SalI and NotI. A 50 μL digestion mixture was prepared as follows: 10 μL plasmid, 1 μL SalI, 1 μL NotI, 5 μL 10× Buffer, and 33 μL ddH2O. The digestion program was: incubation at 37°C for 30 min. The digestion products were recovered using a gel extraction kit after gel electrophoresis. The recovered products were stored at -20°C.

[0063] (3) Ligation and transformation of the target fragment and the enzyme digestion vector

[0064] Using homologous recombinase, the amplified fragment from step (1) was ligated to the digested vector from step (2) to construct ligation systems: pEMOC2-apxIV upstream + apxIV downstream, pEMOC2-apxIC upstream + apxIC downstream, pEMOC2-apxIIC upstream + apxIIC downstream, and pEMOC2-apxIV upstream + Pnap + apxIIIA + apxIV downstream.

[0065] The ligation reaction mixture consisted of 20 μL of the following components: 2 μL vector, 2 μL fragments, 4 μL 5×CE Buffer, 2 μL homologous recombinase, and 8 μL ddH2O. The ligation reaction program was: incubation at 37°C for 35 min.

[0066] The ligation products were transformed into DH5α competent cells. Specifically, 1 μL of the ligation product was added to 50 μL of competent cells, gently mixed by pipetting, and incubated on ice for 30 min. Then, the cells were heat-shocked in a 42°C water bath for 90 s, and immediately cooled on ice for 3 min. 600 μL of LB liquid medium was added, and the cells were incubated in a shaker at 37°C and 180 rpm for 50 min. The cells were then centrifuged at 8000 rpm for 10 min at room temperature, 400 μL of supernatant was discarded, and the cells were resuspended. 100 μL of the bacterial culture was then evenly spread onto LA plates containing 20 μg / mL Cm and incubated at 37°C for 12–15 h.

[0067] Bacteria on the plates were selected and identified using pEMOC2-testF / R primers. Successful construction of recombinant plasmids was confirmed by positive clone identification. The product sizes were: pEMOC2-ΔapxIV (2490bp), pEMOC2-ΔapxIC (2622bp), pEMOC2-ΔapxIIC (2613bp), and pEMOC2-Pnap+apxIIIA (6049bp). See attached figures. Figure 1 A, Figure 1 B.

[0068] 1 μL of recombinant plasmid was chemically transformed into 50 μL of *E. coli* β2155 competent cells, following the same transformation process as above. 100 μL of the bacterial culture was then evenly spread onto LA petri dishes containing 1 mM DAP and 20 μg / mL Cm, and incubated at 37°C for 12-15 h. Single colonies growing on the petri dishes are the donor bacteria.

[0069] 2. Screening of mutant strains

[0070] (1) The pEMOC2-ΔapxIV donor strain was transferred into the recipient strain WH01 via conjugation transfer. (See schematic diagram of gene deletion strain construction.) Figure 2 The first homologous recombination, with a correct single crossover, can amplify both fragments A and B simultaneously. The second homologous recombination, with a correct double crossover, can only amplify fragment A. The specific steps are as follows:

[0071] The donor and recipient bacteria were cultured separately to mid-log phase. 1 mL of each bacterial culture was mixed and centrifuged to collect the bacterial pellet. The pellet was washed three times with 1 mL TSB (containing 10 μg / mL NAD, 1 mM DAP + 1 mM MgSO4). The pellet was then resuspended in 150 μL TSB (containing 10 μg / mL NAD, 1 mM DAP + 1 mM MgSO4) and dropped onto a sterile nitrocellulose membrane. The membrane was then spread evenly on TSA (containing 10 μg / mL NAD, 1 mM DAP + 1 mM MgSO4) and incubated at 37°C for 6 h. The bacteria on the membrane were washed off with physiological saline, diluted 10-fold, and spread onto TSA (containing 10 μg / mL NAD, 2 μg / mL CCM) plates. The plates were incubated at 37°C for 24 h. The resulting colonies were identified and transferred to TSA (containing 10 μg / mL NAD, 2 μg / mL CCM) plates.

[0072] Single crossover identification involved extracting DNA from the conjugated mixed colonies and identifying it using apxIV-UF / DR primers. A single colony amplifying two target bands (1031 bp and 6449 bp) confirmed a single crossover. Results are shown below. Figure 4 Lane A-1, lane 1, indicates a correct single crossover. The 1031bp band corresponds to the internal fragments of the upstream homologous arm (apxIV-up) and downstream homologous arm (apxIV-down) of apxIV, while the 6449bp band corresponds to the internal fragments of the upstream homologous arm (apxIV-up), the apxIV gene, and the downstream homologous arm (apxIV-down) of apxIV. This indicates that the recombinant plasmid has been integrated into the WH01 genome via a single crossover, but a double crossover has not yet been completed.

[0073] Double crossover identification involved culturing single crossover positive colonies, diluting them, and plating them onto TSA (containing 10 μg / mL NAD and 10% sucrose) plates. The presence of sucrose was used to screen for double crossover colonies because the sacB gene causes cell death in sucrose-containing media; only colonies that have undergone double crossover can survive in sucrose-containing media. DNA was extracted from the colonies screened on the TSA plates. Identification was performed using the apxIV-UF / DR primers. A 1031 bp amplification indicates a correct double crossover. The 1031 bp band corresponds to the internal fragments of the upstream homologous arm (apxIV-up) and downstream homologous arm (apxIV-down) of apxIV. The apxIV-inner F / inner R primers were designed for the apxIV gene in the WH01 genome. After double crossover, the apxIV gene is completely replaced, therefore no bands are amplified. Colonies matching the amplification results of both primer pairs are considered correct APPΔapxIV deletion strains. The double crossover identification results are shown in [link to documentation]. Figure 4 A-2 in the middle. Figure 4 Lanes 1-3 of A-2 were identified using apxIV-inner F / inner R primers. Lane 1, with a double-crossover strain genome as the template, did not amplify a band; lane 2, with the maternal strain WH01 genome, amplified a correct 422bp band; lane 3, a negative control, also did not amplify a band. Lanes 4-6 were identified using apxIV-UF / DR primers. Lane 4, with a double-crossover strain genome as the template, amplified a correct 1031bp band; lane 5, with the maternal strain WH01 genome, amplified a correct 6449bp band; lane 6, a negative control, also did not amplify a band. The amplification results of lanes 1 and 4 are consistent with the amplification results of strains lacking the apxIV gene, indicating that the corresponding strains are correct APPΔapxIV deletion strains.

[0074] (2) Then, the pEMOC2-ΔapxIC donor bacteria were transferred into the recipient bacteria APPΔapxIV via conjugation transfer.

[0075] The conjugation transfer method is the same as above. Single exchange identification is performed using apxIC-UF / DR primers. Single exchange is indicated by amplification of two target bands at 541 bp and 1060 bp. See the results below. Figure 4 B-1 in the culture was cultured and passaged after a single crossover. Double crossover identification was performed using apxIC-UF / DR primers, which amplified a 541bp band. Identification with apxIC-inner F / inner R primers failed to amplify any band. Colonies matching the amplification results of both primer pairs were considered the correct APPΔapxIVΔapxIC deletion strain. Results are shown below. Figure 4 B-2 in the middle. Figure 4Lanes 1-3 of B-2 were identified using apxIC-inner F / inner R primers. Lane 1, with a double-crossover strain genome as the template, did not amplify a band; lane 2, with the maternal strain WH01 genome, amplified a correct 441 bp band; lane 3, a negative control, also did not amplify a band. Lanes 4-6 were identified using apxIC-UF / DR primers. Lane 4, with a double-crossover strain genome as the template, amplified a correct 541 bp band; lane 5, with the maternal strain WH01 genome, amplified a correct 1060 bp band; lane 6, a negative control, also did not amplify a band. The amplification results of lanes 1 and 4 are consistent with the amplification results of strains lacking the apxIC gene, indicating that the corresponding strains are the correct APPΔapxIVΔapxIC deletion strains.

[0076] (3) The donor bacterium pEMOC2-ΔapxIIC was transferred into the recipient bacterium APPΔapxIVΔapxIC again via conjugation transfer. The conjugation transfer method was the same as above. Single exchange identification was performed using apxIIC-UF / DR primers. Single exchange was indicated by amplification of two target bands, 577bp and 1060bp. The results are shown in [see attached image]. Figure 4 The C-1 single crossover culture was continued and plated. Double crossover identification was performed using the apxIIC-UF / DR primers, which amplified a 577bp target band. However, the apxIIC-inner F / inner R primers failed to amplify the band. Colonies matching the amplification results of both primer pairs were considered the correct APPΔapxIVΔapxICΔapxIIC deletion strain. Results are shown in […]. Figure 4 C-2 in it. Figure 4 Lanes 1-3 of C-2 were identified using apxIIC-inner F / inner R primers. Lane 1, with a double-crossover strain genome as the template, did not amplify a band; lane 2, with the maternal strain WH01 genome, amplified a correct 436 bp band; lane 3, a negative control, also did not amplify a band. Lanes 4-6 were identified using apxIIC-UF / DR primers. Lane 4, with a double-crossover strain genome as the template, amplified a correct 577 bp band; lane 5, with the maternal strain WH01 genome, amplified a correct 1060 bp band; lane 6, a negative control, also did not amplify a band. The amplification results of lanes 1 and 4 are consistent with the amplification results of strains lacking the apxIIC gene, indicating that the corresponding strains are the correct APPΔapxIVΔapxICΔapxIIC deletion strains.

[0077] (4) Finally, the donor bacterium pEMOC2-Pnap+apxIIIA was transferred into the recipient bacterium APPΔapxICΔapxIICΔapxIV via conjugation transfer. A schematic diagram of the gene insertion strain construction is shown below. Figure 3 .

[0078] The conjugation transfer method is the same as above. Single crossover identification is performed using apxIV-UF / DR primers. Single crossover is indicated by amplification of two target bands at 1031 bp and 4548 bp. See the results below. Figure 4 D-1 cells were cultured and plated after a single crossover. Double crossover identification was performed using apxIV-UF / DR primers, which amplified a 4548bp target band. Using Pnap-inner F / apxIIIA-inner R primers, a 2091bp band was amplified. Colonies matching the amplification results of both primer pairs were considered the correct APPΔapxIVΔapxICΔapxIIC / apxIIIA. + Deletion strains, results are shown in Figure 4 D-2 in the middle. Figure 4 Lanes 1-3 of D-2 were identified using the apxIV-UF / DR primers. Lane 1, with a double-crossover template, amplified a 4548 bp band; lane 2, with the maternal strain WH01's genome, amplified a correct 6449 bp band; lane 3, a negative control, showed no band amplification. Lanes 4-6 were identified using the Pnap-inner F / apxIIIA-inner R primers. Lane 4, with a double-crossover template, amplified a correct 2091 bp band; lane 5, with the maternal strain WH01's genome, showed no band amplification; lane 6, a negative control, also showed no band amplification. The amplification results of lanes 1 and 4 are consistent with the amplification results of strains inserting the Pnap / apxIIIA gene, indicating that the corresponding strain is the correct APPΔapxIVΔapxICΔapxIIC / apxIIIA. + Deletion strain.

[0079] The mutant strain was named WH01-3A. The genome of strain WH01-3A was extracted, and all modified genes in WH01-3A were identified by PCR, with the genome of the maternal strain WH01 serving as a control. The results are shown below. Figure 4The deletion of the apxIV gene and the insertion of the Pnap / apxIIIA gene were identified using primers apxIV-inner F / inner R (lanes 1-3), apxIV-UF / DR (lanes 4-6), and Pnap-inner F / apxIIIA-inner R (lanes 7-9). Lane 1 showed no amplification of the apxIV band in WH01-3A, lane 2 showed a 422bp band amplified in WH01, and lane 3 was the negative control; lane 4 showed a 4548bp band amplified in WH01-3A, lane 5 showed a 6449bp band amplified in WH01, and lane 6 was the negative control; lane 7 showed a 2091bp band amplified in WH01-3A, lane 8 showed no amplification in WH01, and lane 9 was the negative control. This indicates that the apxIV gene was successfully deleted and the Pnap / apxIIIA gene was successfully inserted in strain WH01-3A. The deletion of the apxIC gene was identified using primers apxIC-innerF / innerR (lanes 10-12) and apxIC-UF / DR (lanes 13-15). Lane 10 showed no apxIC band amplified in WH01-3A, lane 11 showed a 441bp band amplified in WH01, and lane 12 was the negative control. Lane 13 showed a 541bp band amplified in WH01-3A, lane 14 showed a 1060bp band amplified in WH01, and lane 15 was the negative control. This indicates that the apxIC gene was successfully deleted in strain WH01-3A. The deletion of the apxIIC gene was identified using primers apxIIC-innerF / innerR (lanes 16-18) and apxIIC-UF / DR (lanes 19-21). Lane 16 showed no apxIIC band amplified in WH01-3A, lane 17 showed a 436bp band amplified in WH01, and lane 18 was the negative control. Lane 19 showed a 577bp band amplified in WH01-3A, lane 20 showed a 1060bp band amplified in WH01, and lane 21 was the negative control. This indicates that the apxIIC gene was successfully deleted in strain WH01-3A.

[0080] Example 2: Detection of biological characteristics of attenuated strain of Actinobacillus pleuropneumoniae type 1 sera

[0081] Comparative analysis of the biological characteristics of the constructed attenuated strain and the parent strain.

[0082] 1. Growth capacity

[0083] Single colonies of WH01 and WH01-3A were picked and inoculated into 5 mL of TSB (containing 10 μg / mL NAD) liquid medium and incubated overnight at 37°C in a shaker. The next day, they were transferred 1:100 to fresh TSB (containing 10 μg / mL NAD) liquid medium and incubated at 37°C in a shaker at 180 rpm. OD was measured at 150 μL of bacterial culture approximately every 2 hours. 600 nm Values ​​were calculated and a growth curve was plotted. The results are as follows: Figure 5 As shown, the attenuated strain WH01-3A and the parent strain WH01 maintained the same growth rate during the mid-log phase, while the attenuated strain WH01-3A grew faster than the parent strain WH01 during the late logarithmic and plateau phases.

[0084] 2. Hemolytic activity

[0085] Single colonies of WH01 and WH01-3A were picked and transferred to blood agar plates, four colonies of each type. The blood agar plates were incubated at 37°C for 12 hours, and the presence of hemolytic zones in the transferred colonies was observed. The results are as follows: Figure 6 As shown, hemolytic zones appeared around all four single colonies at the transformation point of the parent strain WH01, while no hemolytic zones were observed around the single colonies of the deletion strain WH01-3A.

[0086] 3. Stable heritability

[0087] Single colonies of WH01-3A were picked and inoculated into 5 mL of TSB (containing 10 μg / mL NAD) liquid medium and cultured in a shaker at 37°C for 12 h as the first generation. The colonies were then transferred 1:100 to fresh TSB (containing 10 μg / mL NAD) liquid medium and cultured for another 12 h as the second generation. This process was repeated until the 20th generation. Genomic DNA was extracted every two generations, and deleted and inserted genes were identified by PCR to observe the genetic stability of deleted and inserted genes in the attenuated strain. Results are shown below. Figure 7 When the mutant strain WH01-3A was passaged for 20 generations, all the modified genes were stably inherited.

[0088] Example 3: Evaluation of the safety and immunoprotective effect of attenuated strain of Actinobacillus pleuropneumoniae type 1 serum 1. Virulence in mice (LD50) 50 )

[0089] Sixty-six four-week-old female KM mice were randomly divided into 11 groups of six each. Five doses of the maternal strain WH01 and the mutant strain WH01-3A were administered for challenge, and a negative control group was included. Both WH01 and the mutant strain WH01-3A were cultured to mid-logarithmic growth phase, and OD... 600 The concentration is approximately 0.3, at which point the bacterial culture concentration is 1×10⁻⁶. 9 CFU / mL, bacteria were collected by centrifugation at 6000 rpm for 5 min, washed twice with PBS, and the bacterial suspension was diluted to the expected challenge dose for later use. Simultaneously, the suspension was diluted for plate counting. Mice in each group were intraperitoneally infected with 200 μL of the prepared bacterial suspension, while mice in the negative control group were intraperitoneally injected with 200 μL of physiological saline. Mice were observed continuously for 60 h, and the survival rate and LD50 of each group were calculated. 50 .

[0090] The results are shown in Table 2. The LD50 of the mutant strain WH01-3A in mice was... 50Compared to the parent strain WH01, the virulence was increased by about 150 times, meaning the virulence of the mutant strain decreased by about 150 times.

[0091] Table 2. Virulence identification of maternal strain WH01 and mutant strain WH01-3A in mice.

[0092]

[0093] 2. Immunoprotective effect on mice

[0094] When the mutant strain WH01-3A was cultured to mid-log phase, OD 600nm The concentration is approximately 0.3, at which point the bacterial culture concentration is 1×10⁻⁶. 9 CFU / mL, centrifuge at 6000 rpm for 5 min to collect bacteria, wash twice with PBS, and adjust the bacterial concentration to 4 × 10⁻⁶. 9 CFU / mL available for use.

[0095] Seventy-two 6-week-old female Balb / c mice were randomly divided into 9 groups of 8 mice each. Groups 1-3 were immunized with WH01-3A, and groups 4-6 were immunized with Merck's imported subunit vaccine. Mice were divided into two groups: the MSD immunization group (hereinafter referred to as the MSD immunization group) and the negative control group (groups 7-9). Each group of mice received an intramuscular injection of the vaccine or PBS. Fourteen days after the initial immunization, a second immunization was administered at the same dose. Serum samples were collected 14 days after both the initial and second immunizations to detect antibodies against whole-cell antigens of types 1, 5, and 15, as well as antibodies against ApxIA, ApxIIA, and ApxIIIA proteins. Fourteen days after the second immunization, mice were challenged with clinical strains of type 1 (WH01), type 5, and 15, respectively. The immunoprotection rate of each group was calculated after challenge. The immunization groupings are shown in Table 3.

[0096] Table 3 Immunoassay Groups and Doses

[0097]

[0098] Indirect ELISA results show that, Figure 8 As shown, after the second immunization, there were no significant differences in S1, S5, and S15 between the WHO01-3A immunization group and the MSD immunization group. The antibody levels of ApxIA and ApxIIIA in the MSD immunization group were higher than those in the WHO01-3A immunization group, while the antibody levels of ApxIIA in the MSD immunization group were lower than those in the WHO01-3A immunization group.

[0099] Challenge experiments were conducted using APP strain 1 (WH01) and clinical isolates of strains 5 and 15, respectively. All groups exhibited symptoms such as rough, disheveled hair and loss of appetite within 2 hours post-infection. All negative control groups died within 24 hours. The protection rates against strains 1, 5, and 15 in the WH01-3A immunization group were 87.5%, 75%, and 87.5%, respectively. The protection rates against strains 1, 5, and 15 in the MSD immunization group were 50%, 75%, and 87.5%, respectively. These results are as follows: Figure 9 As shown, the attenuated vaccine prepared from strain WH01-3A has better immunization protection against type 1 than the commercially available MSD vaccine, while its immunization protection against types 5 and 15 is consistent. Therefore, strain WH01-3A can be used as a candidate strain for a porcine infectious pleuropneumonia vaccine.

Claims

1. A porcine Actinobacillus pleuropneumoniae serotype 1 attenuated mutant strain, characterized in that, The strain is a porcine Actinobacillus pleuropneumoniae serotype 1 strain that lacks the apxIV, apxIC, and apxIIC genes but contains the Pnap and apxIIIA genes.

2. The attenuated mutant strain of Actinobacillus pleuropneumoniae serotype 1 according to claim 1, characterized in that, It is obtained by constructing using the following method. (1) Using the genome of the clinical strain WH01 of Actinobacillus pleuropneumoniae serotype 1 as a template, the apxIV-up fragment, apxIV-down fragment, apxIC-up fragment, apxIC-down fragment, apxIIC-up fragment, apxIIC-down fragment, and the promoter (Pnap) fragment of the nap gene were amplified from the genome of strain WH01 by PCR. The apxIIIA gene fragment was amplified from the genome of strain JL03. (2) The apxIV-up and apxIV-down fragments were ligated into the pEMOC2 vector to obtain the recombinant plasmid pEMOC2-ΔapxIV; The apxIC-up and apxIC-down fragments were ligated into the pEMOC2 vector to obtain the recombinant plasmid pEMOC2-ΔapxIC; The apxIIC-up and apxIIC-down fragments were ligated into the pEMOC2 vector to obtain the recombinant plasmid pEMOC2-ΔapxIIC; the apxIV-up, Pnap, apxIIIA, and apxIV-down fragments were ligated into the enzyme-digested pEMOC2 vector to obtain the recombinant plasmid pEMOC2-Pnap+apxIIIA. (3) The recombinant plasmids obtained in step (2) were transformed into Escherichia coli β2155 competent cells by chemical transformation, and positive clones were selected for identification. (4) After mixing pEMOC2-ΔapxIV positive clones with WH01, single-exchange positive bacteria were identified. After reverse screening of the sacB gene, the single-exchange positive bacteria were identified by PCR to obtain double-exchange positive bacteria, namely APPΔapxIV strain. (5) After mixing pEMOC2-ΔapxIC positive clones with APPΔapxIV from step (4) for culture, single-exchange positive bacteria were identified. After reverse screening of the sacB gene, the single-exchange positive bacteria were identified by PCR to obtain double-exchange positive bacteria, namely APPΔapxIVΔapxIC strain. (6) After mixing pEMOC2-ΔapxIIC positive clones with the APPΔapxIVΔapxIC strain from step (5) for culture, single-exchange positive bacteria were identified. After reverse screening of the sacB gene, the single-exchange positive bacteria were identified by PCR to obtain double-exchange positive bacteria, namely the APPΔapxIVΔapxICΔapxIIC strain. (7) Finally, the pEMOC2-Pnap+apxIIIA positive clones were mixed with the APPΔapxIVΔapxICΔapxIIC clones from step (6) and cultured to obtain single-exchange positive bacteria. The single-exchange positive bacteria were then subjected to reverse selection using the sacB gene and PCR identification to obtain double-exchange positive bacteria, resulting in APPΔapxIVΔapxICΔapxIIC / apxIIIA. + The strain was identified and named *Actinomyces pleuropneumoniae* serotype 1 attenuated mutant strain WH01-3A.

3. The method according to claim 2, characterized in that, The PCR reaction system in step (1) is as follows: 2×SuperKfx MasterMix 25μL, upstream primer 1μL, downstream primer 1μL, genome 1μL, ddH2O 22μL; the PCR reaction program is: 95℃ pre-denaturation for 5min, 95℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 20s, 30 cycles, 72℃ for 10min. The primers for amplifying the apxIV-up fragment are SEQ ID NO.1 and SEQ ID NO.2, respectively, and the primers for amplifying the apxIV-down fragment are SEQ ID NO.3 and SEQ ID NO.4, respectively. The primers for amplifying the apxIC-up fragment are SEQ ID NO.9 and SEQ ID NO.10, respectively, and the primers for amplifying the apxIC-down fragment are SEQ ID NO.11 and SEQ ID NO.12, respectively. The primers for amplifying the apxIIC-up fragment are SEQ ID NO.17 and SEQ ID NO.18, respectively, and the primers for amplifying the apxIIC-down fragment are SEQ ID NO.19 and SEQ ID NO.20, respectively. The primers for amplifying the promoter (Pnap) fragment of the nap gene are SEQ ID NO.27 and SEQ ID NO.28, respectively, and the primers for amplifying the apxIIIA gene fragment are SEQ ID NO.29 and SEQ ID NO.30, respectively.

4. The method according to claim 3, characterized in that, Step (2) further includes digesting the pEMOC2 empty vector plasmid with restriction endonucleases SalI and NotI. A 50 μL digestion system was prepared: 10 μL plasmid, 1 μL SalI, 1 μL NotI, 5 μL 10× Buffer, and 33 μL ddH2O. The digestion program was: incubation at 37°C for 30 min. Step (2) also includes using homologous recombinase to ligate the amplified fragment from step (1) with the digested pEMOC2 vector; the ligation reaction system is 20 μL: 2 μL vector, 2 μL fragments, 4 μL 5×CE Buffer, 2 μL homologous recombinase, and 8 μL ddH2O; the ligation reaction program is: incubation at 37℃ for 35 min.

5. The method according to claim 4, characterized in that, In step (4), primers as shown in SEQ ID NO.5 and SEQ ID NO.6 are used to identify single-exchange positive bacteria, and primers as shown in SEQ ID NO.5 to SEQ ID NO.8 are used to identify double-exchange positive bacteria APPΔapxIV strain; In step (5), single-exchange positive bacteria are identified using primers shown in SEQ ID NO.13 and SEQ ID NO.14, and double-exchange positive bacteria APPΔapxIVΔapxIC strains are identified using primers shown in SEQ ID NO.13 to SEQ ID NO.

16. In step (6), single-exchange positive bacteria are identified using primers shown in SEQ ID NO.21 and SEQ ID NO.22, and double-exchange positive bacteria APPΔapxIVΔapxICΔapxIIC strains are identified using primers shown in SEQ ID NO.21 to SEQ ID NO.

24. In step (7), single-exchange positive bacteria are identified using primers shown in SEQ ID NO. 5 and SEQ ID NO. 6, and double-exchange positive bacteria APPΔapxIVΔapxICΔapxIIC / apxIIIA are identified using primers shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 31, and SEQ ID NO.

32. + Strains, i.e., the target strain.

6. The use of the attenuated mutant strain of Actinobacillus pleuropneumoniae serotype 1 as described in claim 1 in the preparation of an attenuated live vaccine of Actinobacillus pleuropneumoniae.

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