Antigen combination, porcine contagious pleuropneumonia-Klebsiella hyopneumoniae bivalent vaccine and application thereof

Abstract

The invention is suitable for the technical field of veterinary vaccines, and provides an antigen combination, a porcine contagious pleuropneumonia-klebsiella hyopneumoniae bivalent vaccine and application thereof. The invention develops a bivalent vaccine (APP-KP bivalent vaccine) capable of simultaneously preventing and treating porcine contagious pleuropneumonia and klebsiella hyopneumoniae. The vaccine has obvious effects in the aspects of reducing the morbidity, reducing the body temperature of piglets, reducing the asthma frequency, relieving lung pathological injury and effectively removing actinobacillus pleuropneumoniae (APP) and klebsiella hyopneumoniae (KP) in vivo. Through combination of TbpB-C and AdhE protein antigens of actinobacillus pleuropneumoniae and JLKP3 and JLKP9 inactivated bacterium antigens of klebsiella hyopneumoniae, powerful support is provided for healthy development of the pig industry.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccines, and in particular relates to an antigen combination, a porcine contagious pleuropneumonia-swine Klebsiella pneumoniae bivalent vaccine and applications thereof. Background Art

[0002] Porcine contagious pleuropneumoniae (PCP), also known as necrotizing pleuropneumonia, is a highly contagious and fatal respiratory infectious disease caused by Actinobacillus pleuropneumoniae (APP). Pigs of all ages are susceptible to this disease, which is common in growing pigs and adult pigs. It is mainly characterized by acute hemorrhagic fibrinous pneumonia and chronic fibrinous necrotizing pleurisy. The mortality rate of the most acute type can reach 80-100%, posing a serious threat to the breeding industry. In recent years, the clinical isolation rate of APP has been increasing year by year. It often co-infects with other pathogens such as Haemophilus parasuis, Pasteurella multocida, Klebsiella pneumoniae, PCV2 and PRRSV, causing porcine respiratory syndrome, weakening the lung defense function of pigs, and increasing the difficulty of prevention and treatment.

[0003] Actinobacillus pleuropneumoniae belongs to the family Pasteurellaceae and the genus Actinomycetes. It can be divided into 19 serotypes based on its capsular polysaccharide, and there is a lack of cross-protection between the serotypes. The serotypes of APP prevalent in different regions of my country are different. The latest epidemiological survey shows that types 1, 3, 5, 7 and 15 are the dominant serotypes in my country. Bioinformatics studies have revealed that TbpB (transferase) and AdhE (acetaldehyde / alcohol dehydrogenase) are highly conserved among different serotypes of APP and are important virulence factors of APP. TbpB-C and AdhE are also important antigens for PCP vaccines. Vaccines prepared with these two antigens can provide complete protection against APP type 1 and APP type 3 infections in mice (Chinese patent with publication number CN202411212848.0).

[0004] Klebsiella pneumoniae is one of the important conditional pathogens, which has a serious impact on human and animal health. In recent years, Klebsiella pneumoniae has gradually become a conditional pathogen second only to Escherichia coli. It is widely distributed in the natural environment, and can cause secondary infection in various parts of the animal body, causing pneumonia, mastitis, sepsis and other suppurative inflammations. Klebsiella pneumoniae can be divided into 78 serotypes according to the capsule type. The K1 and K2 types are the most harmful and prevalent in hospitals, but there is no relevant data to verify it in pigs. A serological epidemiological survey of pig herds in Northeast China and Shandong Province showed that the infection rate of Klebsiella pneumoniae was 27.28%, which poses a huge potential threat to the pig farming industry. In addition, since there is no commercial vaccine for this disease on the market, the abuse of antibiotics in animal farming has led to an increasingly serious problem of drug resistance in Klebsiella pneumoniae.

[0005] In view of the serious harm of porcine contagious pleuropneumonia, the inability of existing vaccines to provide good cross-protection effects, and the current situation that there is no commercial vaccine for porcine Klebsiella pneumoniae, the present invention proposes an antigen combination, a porcine contagious pleuropneumonia-porcine Klebsiella pneumoniae bivalent vaccine and its application. Summary of the invention

[0006] The purpose of the present invention is to provide an antigen combination, a porcine contagious pleuropneumonia-swine Klebsiella pneumoniae bivalent vaccine and its application, aiming to solve the problems raised in the above background technology.

[0007] The purpose of the present invention is achieved through the following technical solutions: An antigen combination of Actinobacillus pleuropneumoniae and Klebsiella suiconiae, comprising AdhE and TbpB-C proteins of Actinobacillus pleuropneumoniae and inactivated bacteria JLKP3 and JLKP9 of Klebsiella suiconiae; the JLKP3 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M 2025116; the JLKP9 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M 2025117.

[0008] An application of the antigen combination of Actinobacillus pleuropneumoniae and Klebsiella hyopneumoniae described above in the preparation of a combined vaccine for porcine contagious pleuropneumoniae and Klebsiella hyopneumoniae.

[0009] A porcine contagious pleuropneumonia-swine Klebsiella pneumoniae combined vaccine comprises the antigen combination of Actinobacillus pleuropneumoniae and Klebsiella swine pneumonia as described above, and MONTANIDE™ Gel 01 adjuvant.

[0010] Furthermore, the volume ratio of the MONTANIDE™ Gel 01 adjuvant to the protein and inactivated bacteria mixture is 1:9.

[0011] A use of the above-mentioned porcine contagious pleuropneumonia-swine Klebsiella pneumoniae bivalent vaccine in the preparation of a drug for preventing and treating porcine contagious pleuropneumonia and swine Klebsiella pneumoniae.

[0012] The invention discloses an application of JLKP3 and JLKP9 inactivated bacteria in preparing a vaccine for Klebsiella pneumoniae in pigs.

[0013] A vaccine for porcine Klebsiella pneumoniae, comprising inactivated bacteria JLKP3 and JLKP9.

[0014] A use of the above-mentioned Klebsiella pneumoniae vaccine in the preparation of a medicine for preventing and treating Klebsiella pneumoniae.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention has developed a bivalent vaccine (APP-KP bivalent vaccine) that can simultaneously prevent and treat porcine infectious pleuropneumonia and porcine Klebsiella pneumoniae. The vaccine has shown significant effects in reducing piglet body temperature, reducing panting frequency, alleviating lung pathological damage, and effectively clearing Actinobacillus pleuropneumoniae (APP) and Klebsiella suis pneumoniae (KP) in the body. By combining the TbpB-C and AdhE protein antigens of Actinobacillus pleuropneumoniae and the JLKP3 and JLKP9 inactivated bacterial antigens of Klebsiella suis pneumoniae, the present invention provides strong support for the healthy development of the pig farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The expression of candidate proteins, where M: Protein Marker; 1: before induction; 2-3: supernatant and precipitate of recombinant plasmid expression after IPTG induction; 4: purified recombinant protein.

[0017] Figure 2 The figure is for the screening of candidate antigens of Klebsiella pneumoniae; A is the survival rate of mice in each immunization group after infection; B is the clinical symptom score of mice in each group after infection; C is the antibody titer in the serum of each group 28 days after immunization; D is the bacterial load in the lungs, liver and spleen of each group of mice 72 hours after infection.

[0018] Figure 3 Evaluation of the immune effect of APP-KP bivalent vaccine on mice; A is the survival rate of APP1-infected mice after bivalent vaccine immunization; B is the survival rate of APP3-infected mice after bivalent vaccine immunization; C is the survival rate of JLKP3-infected mice after bivalent vaccine immunization; D is the survival rate of JLKP9-infected mice after bivalent vaccine immunization; E is the TbpB-C antibody titer in the serum of each group of mice; F is the AdhE antibody titer in the serum of each group of mice; G is the JLKP3 antibody titer in the serum of each group of mice; H is the JLKP9 antibody titer in the serum of each group of mice.

[0019] Figure 4 To evaluate the immunization effect of piglets with the combined vaccine against porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae; A is the monitoring of piglet antibody titer after immunization; B is the monitoring of piglet body temperature after infection; C is the observation of pathological changes in piglet genitals and pathological sections; D is the detection of bacterial load in each tissue. DETAILED DESCRIPTION

[0020] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.

[0021] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0022] Example 1: Protein expression and purification; 1.1 Construction of recombinant expression plasmid; The target fragment was amplified by PCR using the primers described in Table 1, using the genomic DNA of APP of serotype 1 as a template.

[0023]

[0024] After PCR amplification of the target gene, it was subjected to agarose gel electrophoresis and purified by a gel recovery kit. The purified target gene and pET-32a plasmid were double-digested with BamHI and XhoI, respectively. The target gene after digestion was connected to the vector, mixed evenly and placed in a 16°C low-temperature water bath for connection overnight. Finally, the connected recombinant plasmid was transformed into Escherichia coli DH5α competent cells by a kit, and the recombinant expression plasmids peT32a-TbpB-C and peT32a-AdhE were finally constructed. These recombinant expression plasmids were identified by enzyme digestion and sequencing, and they were consistent with expectations, with no sequence mutations and successful insertion into the pET32a vector.

[0025] Among them, the amino acid sequence of TbpB-C protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding TbpB-C protein is shown in SEQ ID NO.2; the amino acid sequence of AdhE protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding AdhE protein is shown in SEQ ID NO.4.

[0026] 1.2 Protein expression and purification; The constructed recombinant plasmid was transformed into competent E. coli BL21 (DE3). The next day, a single colony was picked from the plate and inoculated into LB liquid medium containing Amp+ resistance. The culture was cultured at 37°C and 180 rpm until OD600nm = 0.6-0.8. 200 μl IPTG with a final concentration of 1 mM was added at 16°C to induce expression for 16 hours. The cells were centrifuged at 4°C and 6000 rpm for 10 min, the supernatant was discarded, and the cells were collected. PBS was added to resuspend and ultrasonically disrupt the cells, and the supernatant and precipitate were collected separately.

[0027] The supernatant collected after ultrasound was purified by nickel column affinity chromatography. After the bacteria were ultrasonically disrupted, they were centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was collected, and the pH was adjusted to 8.0 for purification. For specific operation steps, please refer to the Cytiva Ni column purification instructions. Finally, 5×SDS gel loading buffer was added to the 500mM imidazole eluent, and 12% SDS-PAGE was performed after boiling for 10 min to detect the protein expression in different parts. The results showed that the two proteins were expressed in large quantities in the supernatant, and the sizes of TbpB-C and AdhE were 41.76kDa and 114.2kDa, respectively ( Figure 1 ).

[0028] Example 2: Screening of Klebsiella hyopneumoniae antigens; 2.1 Inactivation of candidate antigens; Eight candidate Klebsiella pneumoniae strains (JLKP3, JLKP9, KPP19, KPP17, KPP15, KP69, K36, and KP26) were placed in LB liquid medium and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 ≈ 1.8), formaldehyde was added to the final concentration of 0.2%, the bacterial solution was inactivated at 37℃ for 48h, 200μL was spread on LB plate to test the inactivation effect, appropriate amount of bacteria was taken, centrifuged and resuspended for later use.

[0029] Among them, Klebsiella pneumoniae KPP19, KPP17, KPP15, KP69, K36 and KP26 were isolated from the laboratory and can be used.

[0030] Klebsiella pneumoniae JLKP3 is deposited in China Center for Type Culture Collection, the address of the depository is Wuhan University, Wuhan, China, the postal code is 430072, the deposit date is January 13, 2025, the deposit number is CCTCC NO: M2025116, and the classification name is Klebsiella Pneumoniae JLKP3.

[0031] Klebsiella pneumoniae JLKP9 is deposited in China Center for Type Culture Collection, the address of the depository is Wuhan University, Wuhan, China, the postal code is 430072, the deposit date is January 13, 2025, the deposit number is CCTCC NO: M2025117, and the classification name is Klebsiella Pneumoniae JLKP9.

[0032] 2.2 Evaluation of the immune protection effect of Klebsiella hyopneumoniae antigen in mice; Take the inactivated bacteria prepared in 2.1 and mix them with MONTANIDE™ Gel 01 adjuvant (v / v=9:1) to make an inactivated vaccine. The dose of each antigen is about 3*10 9 CFU. 50 SPF-grade 6-week-old female ICR mice were selected, 5 mice in each group, and immunized according to the experimental scheme in Table 2:

[0033] A second immunization was performed 14 days after the first immunization, and a virus attack was performed 14 days after the second immunization. Blood was collected from the tail vein of each group of mice before the first immunization, the second immunization and the virus attack, and the serum was packaged and frozen for later use. The mice were observed for 5 consecutive days after the virus attack, and the survival of the mice was counted. 72 hours after infection, the lungs (Lung), livers (Liver) and spleens (Spleen) of 3 mice in each group were aseptically collected, ground into homogenate, and diluted with PBS to a uniform multiple. 100μL was spread on the LB plate, and the number of plates was counted after 24 hours. The survival rate of mice in each group after the virus attack showed ( Figure 2 Middle A), all mice in the immunized groups survived after infection, especially the mice in the JLKP3 and JLKP9 groups had the best clinical status ( Figure 2 The antibody titer results of the serum of each group at 28 days after immunization showed ( Figure 2 In the middle C, the specific antibody titers in the sera of the JLKP3, JLKP9, KPP19, KPP17, KPP15, KP69, K36 and KP26 groups were significantly higher than those in the adjuvant control group and PBS group. The bacterial load in the mouse tissues (lungs, liver and spleen) of each group at 72h after infection showed ( Figure 2 In the middle (D), the JLKP3 and JLKP9 groups had the lowest bacterial load in tissues and organs and the best clinical status. Combining the survival rate, specific antibody titer, tissue bacterial load and bacterial load data, the inactivated JLKP3 and JLKP9 bacteria were determined to be candidate antigens for the vaccine against Klebsiella pneumoniae in swine.

[0034] Example 3: Preparation of a combined vaccine against porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae and evaluation of its immune effect in mice; 3.1 Preparation of the combined vaccine for porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae; Preparation process: Take the TbpB-C and AdhE proteins in Example 1 and the inactivated bacteria JLKP3 and JLKP9 in Example 2 and mix them thoroughly, dilute them appropriately according to their concentrations and mix them evenly, mix the diluted proteins and inactivated bacteria with MONTANIDE™ Gel 01 adjuvant (protein and inactivated bacteria: MONTANIDE™ Gel 01 adjuvant = 9:1 (v / v)), and after full combination, obtain the APP-KP bivalent vaccine.

[0035] The volume of each APP-KP dual vaccine is 200 μL, containing 66 μg TbpB-C protein, 33 μg AdhE protein, 3*10 9 CFU JLKP3 inactivated bacteria, 3*10 9 CFU JLKP9 inactivated bacteria and adjuvant.

[0036] 3.2 Evaluation of the immune effect of the combined vaccine against porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae in mice; 60 SPF female ICR mice aged 6 weeks were selected, 5 mice in each group, and immunized according to the experimental scheme in Table 3:

[0037] The second vaccination was carried out 14 days after the first vaccination, and the virus was challenged 14 days after the second vaccination. Blood was collected from the tail vein of each group of mice before the first and second vaccinations and the virus was challenged, and the serum was packaged and frozen for later use. The results showed that the APP-KP bivalent vaccine could provide 80% (4 / 5) protection against both APP1 and APP3 infections ( Figure 3 After infection with JLKP3 and JLKP9 strains, all mice in the PBS group and adjuvant control group died, while the survival rate of the vaccine group was 100% (5 / 5) ( Figure 3 Medium C) and 80% (4 / 5) ( Figure 3 The serum antibody titer results showed that the levels of TbpB-C, AdhE, JLKP3 and JLKP9 specific antibodies in mice increased significantly after the first immunization, and increased again after the second immunization, while the levels of each specific antibody in the PBS group and adjuvant control group did not change significantly compared with before immunization ( Figure 3 The above results prove that the APP-KP bivalent vaccine can stimulate mice to produce high-level specific antibodies against the four antigens TbpB-C, AdhE, JLKP3 and JLKP9, and protect mice from multiple serotypes of APP and KP infection.

[0038] Example 4: Preparation of a combined vaccine for porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae and evaluation of its immune effect on piglets; 4.1 Preparation of the combined vaccine for porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae; The preparation process is the same as that in 3.1 above.

[0039] Each APP-KP bivalent vaccine has a volume of 2 ml and contains 1 mg TbpB-C protein, 0.5 mg AdhE protein, 1*10 10 CFU JLKP3 inactivated bacteria and 1*10 10 CFU JLKP9 inactivated bacteria.

[0040] 4.2 Evaluation of the immune efficacy of the combined porcine contagious pleuropneumonia-swine Klebsiella pneumoniae vaccine in piglets; Twenty-two SPF piglets aged 4 weeks were selected and immunized according to the experimental scheme in Table 4:

[0041] The second immunization was carried out 21 days after the first immunization, and the virus was challenged 14 days after the second immunization. At the same time, blood was collected from the tail vein of each group of mice before the first immunization, the second immunization and the virus challenge, and the serum was packaged and frozen for later use.

[0042] The incidence rate, respiratory rate, LLS lung consolidation score and pathological section score of each group of piglets are shown in Table 5:

[0043] The results showed that after the initial and booster immunization, the specific antibody titers of TbpB-C, AdhE and JLKP3 in the serum of piglets increased ( Figure 4 Middle A). APP1 and JLKP3 were infected 14 days after the second immunization. At the same time, APP commercial vaccine was introduced as a control. There is no commercial vaccine for Klebsiella pneumoniae. After infection, the piglets in the APP control group showed clinical symptoms such as panting (higher respiratory rate), coughing and increased body temperature ( Figure 4 B and Table 5), no respiratory symptoms were found in the APP commercial vaccine group and the APP-KP bivalent vaccine group (APP1), and there were no abnormalities in mental state and activity, and appetite was normal. After the piglets were slaughtered, it was found that the lungs of the APP control group were mostly purple-red, the lungs were enlarged, some tissues were necrotic, there were purulent foci in the lungs, and there were emphysema. The APP commercial vaccine group and the APP-KP bivalent vaccine group (APP1) only had bleeding and surface fibrinous exudates, and the degree of lung consolidation was much lower than that of the APP control group. The KP control group showed bleeding as the main change, with a large number of millet-sized hemorrhages scattered and accompanied by swelling, and substantial areas were visible on the cross-section. The results of pathological tissue sections showed that compared with the APP control group and the KP control group, the APP-KP bivalent vaccine group (APP1 and JLKP3) and the APP commercial vaccine group had fewer inflammatory cell infiltrations in the lungs, better alveolar integrity, and lower bleeding (blood cell deposition) ( Figure 4In addition, a large amount of APP and KP were still present in the tissues 14 days after infection. Compared with the APP control group and the KP control group, the APP-KP bivalent vaccine group reduced the colonization of APP and KP in the liver and lungs, and the amount of APP in the spleen also decreased significantly. The effect of reducing KP in the blood was even more obvious ( Figure 4 (D) demonstrates the important role of APP-KP bivalent vaccine in clearing bacteria in the body.

[0044] In summary, the APP-KP bivalent vaccine plays an important role in reducing piglet fever, reducing the frequency of wheezing, alleviating lung pathological changes, and clearing APP and KP in the body. It prevents various respiratory symptoms after APP and KP infection and is a candidate vaccine for APP and KP.

[0045] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. An antigen combination of Actinobacillus pleuropneumoniae and Klebsiella hyopneumoniae, characterized in that: Including AdhE and TbpB-C proteins of Actinobacillus pleuropneumoniae and inactivated bacteria JLKP3 and JLKP9 of Klebsiella hyopneumoniae; the JLKP3 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M 2025116; the JLKP9 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M2025117.

2. Use of the antigen combination of Actinobacillus pleuropneumoniae and Klebsiella hyopneumoniae according to claim 1 in the preparation of a combined vaccine for porcine contagious pleuropneumoniae and Klebsiella hyopneumoniae.

3. A porcine contagious pleuropneumonia-swine Klebsiella pneumoniae bivalent vaccine, comprising the antigen combination of Actinobacillus pleuropneumoniae and Klebsiella swine pneumonia as claimed in claim 1, and MONTANIDE™ Gel 01 adjuvant.

4. The porcine contagious pleuropneumonia-swine Klebsiella pneumoniae bivalent vaccine according to claim 3, characterized in that: The volume ratio of the MONTANIDE™ Gel 01 adjuvant to the protein and inactivated bacteria mixture is 1:

9.

5. Use of the combined vaccine of porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae according to claim 3 or 4 in the preparation of a medicament for preventing and treating porcine contagious pleuropneumonia and porcine Klebsiella pneumoniae.

6. Use of JLKP3 and JLKP9 inactivated bacteria in the preparation of porcine Klebsiella pneumoniae vaccine.

7. A vaccine for Klebsiella pneumoniae, characterized in that: Contains inactivated bacteria of JLKP3 and JLKP9.

8. Use of the Klebsiella pneumoniae vaccine according to claim 7 in the preparation of a medicament for preventing and treating Klebsiella pneumoniae.

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