Inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae and its application

Abstract

The present invention belongs to the field of biotechnology and veterinary vaccines, and specifically relates to an inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae and its application. The vaccine comprises: rApxⅠ protein shown in SEQ ID NO.1, rApxⅡ protein shown in SEQ ID NO.2 and inactivated porcine Actinobacillus pleuropneumoniae, and the deposit number of the porcine Actinobacillus pleuropneumoniae is CCTCC NO: M20231360. The present invention combines soluble expressed ApxⅠA and ApxⅡA with porcine Actinobacillus pleuropneumoniae serotype 1 inactivated whole bacteria, which can provide cross protection for porcine infectious Actinobacillus pleuropneumoniae of different serotypes. Compared with immunization with APP2022116 alone, the combined vaccine of the present invention improves resistance to porcine Actinobacillus pleuropneumoniae serotype 1 strains, and the protection rate against APP1 type and APP7 type strains is 100%.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and veterinary vaccines, and specifically relates to an inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae and its application. Background Art

[0002] Porcine contagious pleuropneumonia (PCP), caused by Actinobacillus pleuropneumoniae (APP), is a serious infectious disease of pigs, often with acute outbreaks and high mortality, which has brought great harm to the pig industry worldwide (Nahar et al., 2021). The disease is highly contagious and clinically characterized by hemorrhagic, fibrotic and necrotic pneumonia. APP is a Gram-negative short rod with significant pleomorphism and bipolar coloration. The bacteria have capsules, no spores, are non-motile, and are facultative anaerobic bacteria. The bacteria can be divided into two biotypes: biotype I and biotype II based on whether nicotinamide adenine dinucleoside is required during growth. Based on the differences in APP capsule antigens and lipopolysaccharides, biotype I can be divided into 17 serotypes, and biotype II contains two serotypes. The serotypes that have been isolated in my country are 1, 2, 3, 4, 5, 7, 8, 9, and 10. There are many serotypes, among which serotypes 1, 2, 3, and 7 are the main epidemic serotypes (Zhu Xiugao and Li Yanqing, 2017). Due to the rapid course of the disease, the serious problem of drug resistance of pathogens, and the poor effect of antibiotic treatment, as well as the introduction of my country's policy of reducing and prohibiting antibiotics, vaccination is one of the important means of preventing and controlling the disease.

[0003] Since there are many APP serotypes, there is no good cross-reaction between the types (Yao Jiancong et al., 2003). At the same time, the dominant serotypes prevalent in different regions will change over time, which will also increase the difficulty of vaccine development. The whole-bacterial inactivated vaccine is the most effective first-generation vaccine for preventing this disease, but this vaccine can only protect against the same serotype and cannot provide cross-protection (Kim et al., 2011). Due to regulations and biosafety issues, home-grown seedlings are limited to farms infected with this disease, which has limitations in disease prevention and control. Live attenuated vaccines can provide immune protection for immunized animals, but they may restore virulence and return to strong toxins, which poses certain risks and has not yet been widely used (Zhou et al., 2016).

[0004] For porcine contagious pleuropneumonia, the existing vaccines in China are almost all inactivated vaccines. However, the low content of exotoxin components secreted by APP in inactivated vaccines is also the reason for the poor immune effect. Therefore, the research on exotoxins has become the research focus of this disease. Inactivated vaccines such as Keqian Biological's Kefeining, whose antigens include APP types 1, 2, and 7, have high bacterial antigen concentrations and large side effects. There are also some subunit vaccines, such as Merck's subunit vaccine AIPK, but its vaccine is expensive, which increases the cost of breeding. At the same time, pure subunit vaccines lack general protection due to the lack of cell wall components such as LPS.

[0005] Hemolytic exotoxin (Apx) is one of the main virulence factors of APP. Apx toxins are ApxI, ApxII, ApxIII and ApxIV (Frey et al., 1991; Hathroubi et al., 2018). Except for ApxIV toxin, which exists in all serotypes, the other three are only synthesized and secreted by certain serotypes. Serotypes 1, 5, 9, and 11 secrete ApxⅠ and ApxⅡ, serotypes 2, 3, 6, and 8 secrete ApxⅡ and ApxⅢ, type 10 secretes ApxⅠ, and types 7 and 12 secrete ApxⅡ. Apx toxin is not only the main virulence factor of the bacteria, but also the main protective antigen of APP (Ito and Sueyoshi, 2015).

[0006] In the past 30 years, scholars at home and abroad have been committed to exploring the effects of Apx toxin on immunity and APP protection. As early as 1998, there was a literature pointing out that subunit vaccines containing Apx toxin can resist APP9 infection (Chiers et al., 1998); in 2006, Chinese scientists used prokaryotic exogenous expression of rApxⅠ and rApxⅡ, and proved that they had good immune protection effects in the immunization of mice and rabbits (Wang Chunlai et al., 2006; Yan Kexia et al., 2006); in the past 15 years, a large number of graduate students from domestic universities have made significant progress in the study of Apx toxin, and their research results show that Apx toxin has a good immune effect, but without exception, in their studies, whether Apx toxin is truncated or expressed in full length, the expressed Apx toxin is in inclusion bodies, and it needs to be denatured and purified from inclusion bodies (Hu Huican, 2019; Li Peng, 2017; Long Jian, 2020; Song Dan et al. ... al., 2019; Tian Jin, 2019; Wu Dong, 2014; Yu Guangyong, 2008; Zhou Jiaqiang, 2013), or by culturing APP in large quantities in vitro, recovering its culture supernatant and then purifying it (Hua Fanggen, 2012; Ma Yanfang, 2009). Both methods have obvious disadvantages, with low yields and are not suitable for large-scale production. At the same time, the spatial conformation of the protein purified by inclusion bodies may change, resulting in changes in its antigenic determinants and reducing some immunogenicity. Some scholars also use truncated Apx toxins as surface display antigens, express them on the surface of brewer's yeast, and administer them orally to achieve immunity of pigs against APP (Shin et al., 2013). The purpose of these large amounts of work is to obtain Apx toxins and immunize pigs to obtain better protection effects.

[0007] In subunit vaccines, complete proteins usually have better immune effects. This is because the complete protein contains all antigenic determinants and can stimulate a wider immune response. In contrast, truncated proteins only contain some antigenic determinants and may not stimulate the same broad immune response. In the above study, although it was proved that Apx toxin has a good immune effect, it was not possible to express it completely and purify it through better means.

[0008] In view of the above problems, the present invention successfully exogenously expressed the full-length ApxI and ApxⅡ. The protein is highly soluble and expressed in the supernatant. It is mixed with the screened APP1 type highly virulent strain to prepare an inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae. The vaccine can provide more and richer cell wall antigens, and can also induce a strong cell-mediated immune response through ApxⅠ and ApxⅡ, thereby achieving the protection against multiple serotypes of App, and providing a comprehensive solution for the immune prevention and control of porcine contagious pleuropneumoniae. Summary of the invention

[0009] The object of the present invention is to provide an inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae, which comprises two recombinant fusion proteins and inactivated porcine Actinobacillus pleuropneumoniae APP2022116, which are rApxⅠ (amino acid sequence is SEQ ID NO.1), rApxⅡ (amino acid sequence is SEQ ID NO.2) and porcine Actinobacillus pleuropneumoniae with a deposit number of CCTCC NO: M20231360.

[0010] Another object of the present invention is to provide the use of the inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae in the preparation of porcine Actinobacillus pleuropneumoniae vaccine.

[0011] In order to achieve the above invention objectives, the present invention provides the following technical solutions:

[0012] Through virulence testing, the applicant screened out a serotype 1 porcine pleuropneumoniae Actinobacillus from more than 100 strains. The strain has strong virulence and good immunogenicity. It also has cross-protection against the domestically prevalent serotypes 1 and 7 porcine pleuropneumoniae Actinobacillus. The protection rates against APP1 and APP7 strains are 80% and 100%, respectively.

[0013] The applicant sent the strain to the China Center for Type Culture Collection for preservation on July 21, 2023, with classification name: Actinobacillus pleuropneumoniae APP2022116, preservation number: CCTCC NO: M20231360, address: Wuhan University, Wuhan, China.

[0014] The culture and morphological characteristics of the strain are as follows:

[0015] The cell morphology of strain APP2022116 is club-shaped, sometimes linear or pleomorphic, without flagella, non-motile, with a diameter of about 0.5nm to 2.0nm and a length of about 60nm to 450nm. It has high nutritional requirements during cultivation and needs to grow in a TSA plate containing 10% newborn calf serum and 0.01% NAD in an incubator at 37°C. It will form round colonies with complete and smooth edges and light blue fluorescence.

[0016] The protection scope of the present invention includes:

[0017] The invention discloses an inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae, which comprises: rApxⅠ protein shown in SEQ ID NO.1, rApxⅡ protein shown in SEQ ID NO.2 and inactivated type 1 porcine Actinobacillus pleuropneumoniae, and the deposit number of type 1 porcine Actinobacillus pleuropneumoniae is CCTCC NO: M20231360.

[0018] The vaccine described above is preferably: the preparation method of the rApxⅠ protein or rApxⅡ protein comprises:

[0019] The polynucleotide encoding the protein shown in SEQ ID NO.1 or SEQ ID NO.2 is connected to the vector pCold-TF to obtain a recombinant vector, which is then introduced into E. coli BL21 (DE3) for induced fermentation, and the corresponding protein is obtained in the fermentation supernatant;

[0020] In the above-mentioned preparation method, preferably, the polynucleotide encoding the protein of SEQ ID NO.1 is shown as SEQ ID NO.3; the polynucleotide encoding the protein of SEQ ID NO.2 is shown as SEQ ID NO.4.

[0021] In the above-mentioned preparation method, preferably, the fermentation induction expression condition is 16° C., IPTG is added with a final concentration of 1 mM, and the expression is induced at 170 rpm for 24 hours.

[0022] In the above-mentioned preparation method, preferably, after obtaining the fermentation supernatant, the protein is separated and purified according to the following method:

[0023] The pH value of the loading buffer was adjusted to 11, and the sample was loaded using a prepacked column containing a large-pore anion exchange filler Rigose-DEAE through an AKTA pure25 protein chromatography purification system. The loading flow rate was 1 mL / min. During the loading process, the impurities in the supernatant combined with the anion exchange filler, and the target protein was retained in the loading flow-through. After collecting the flow-through, a 10000 MWCO ultrafiltration tube was used to obtain ApxIA or ApxIIA protein by ultrafiltration concentration.

[0024] The protection scope of the present invention also includes: the use of the inactivated subunit combined vaccine of Actinobacillus pleuropneumoniae in the preparation of porcine Actinobacillus pleuropneumoniae vaccine, wherein the application includes preparing the porcine Actinobacillus pleuropneumoniae vaccine with the inactivated subunit combined vaccine of Actinobacillus pleuropneumoniae as the only effective ingredient or one of the effective ingredients.

[0025] Application of inactivated subunit combined vaccine of Actinobacillus pleuropneumoniae in preparing medicine for treating or preventing porcine Actinobacillus pleuropneumoniae infection.

[0026] In the above application, preferably, the Actinobacillus pleuropneumoniae is Actinobacillus pleuropneumoniae serotype 1 and / or 7.

[0027] In the above-mentioned application, preferably, the vaccine dosage form is any dosage form in the prior art.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention uses E. coli prokaryotes to express the full-length exotoxins ApxⅠA and ApxⅡA of porcine Actinobacillus pleuropneumoniae. The two proteins in the present invention have been successfully optimized to achieve soluble expression. At the same time, the applicant has established a method for separating and purifying the protein, which is easy to mass produce and reduces production costs.

[0030] (2) In the present invention, a serotype 1 porcine Actinobacillus pleuropneumoniae strain was screened out from more than 100 strains. It has strong virulence and good immunogenicity. It also has a cross-protection effect against the domestically prevalent serotypes 1 and 7 porcine Actinobacillus pleuropneumoniae. The protection rates against APP1 and APP7 strains are 80% and 100%, respectively.

[0031] (3) The present invention combines soluble expressed ApxⅠA and ApxⅡA with inactivated whole bacteria of porcine Actinobacillus pleuropneumoniae serotype 1, which can provide cross protection against different serotypes of porcine contagious Actinobacillus pleuropneumoniae. Compared with immunization with APP2022116 alone, the combined vaccine of the present invention improves resistance to porcine Actinobacillus pleuropneumoniae serotype 1 strains, and the protection rate against APP1 and APP7 strains is 100%.

[0032] (4) The present invention lays a foundation for the development of an efficient, low-cost, broad-spectrum porcine contagious pleuropneumonia vaccine and provides an effective technical means for the prevention and treatment of porcine contagious pleuropneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of lung autopsy scoring for APP strain virulence screening.

[0034] Figure 2For the purified and recovered ApxⅠ and ApxⅡ proteins.

[0035] Figure 3 Flow chart for animal immunization.

[0036] Figure 4 This is the detection graph of specific antibody levels in the serum of the control group (NC) and the immune group (APP+rApx1+rApx2).

[0037] Figure 5 This is the survival curve of each group of pigs after virus attack.

[0038] Figure 6 This is a comparison of the autopsy results of the control group and the immunized group after APP1 type virus attack.

[0039] Figure 7 This is a comparison of the autopsy results of the control group and the immune group after APP7 virus attack. DETAILED DESCRIPTION

[0040] In order to make the present invention easier to understand, the embodiments of the present invention will be further described below. The present invention is further described and demonstrated in conjunction with the implementation. However, this embodiment is not a limitation of the present invention. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art; the reagents or materials shown, unless otherwise specified, are all from commercial channels.

[0041] The pCold-TF vector was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd. with the catalog number of Code No. 3365.

[0042] Embodiment 1:

[0043] Screening of Actinobacillus pleuropneumoniae APP2022116 and its immunogenicity: 1. Screening of Actinobacillus pleuropneumoniae APP2022116

[0044] In 2021-2022, the diseased pigs collected from large-scale pig farms across the country were inoculated on TSA plates containing 10% newborn calf serum and 0.01% NAD under aseptic operation and incubated at 37°C overnight. The isolated colonies were identified by PCR. The strains confirmed by PCR to be porcine Actinobacillus pleuropneumoniae were screened. After preliminary screening, 35 strains of porcine Actinobacillus pleuropneumoniae with serotype 1 were selected for the next animal test to compare virulence.

[0045] One hundred and eighty four-week-old female KM mice were randomly divided into 36 groups, with 5 mice in each group. Five mice were used as negative control and injected with normal saline. The other groups were intraperitoneally injected with 5×10 of the above 35 strains of Actinobacillus pleuropneumoniae. 7 / mouse, and observed for 7 consecutive days. The morbidity and mortality of the mice were observed and recorded. The results are shown in Table 1. According to the virulence conditions in Table 1, 20 strains with stronger virulence were further screened, and the challenge dose was reduced for the second screening.

[0046] Table 1 Results of the first toxicity comparison

[0047]

[0048]

[0049] 210 4-week-old female KM mice were randomly divided into 21 groups, with 10 mice in each group. Ten mice were taken as negative control. The other groups were intraperitoneally injected with 1×10 7 / mouse, and observed for 7 consecutive days. The morbidity and mortality of mice were observed and recorded. The results are shown in Table 2. According to the virulence conditions in Table 2 and the comparison with the clinical symptoms of mouse death, 5 strains of bacteria, 1-9, 1-16, 1-20, 1-26, and APP-2, were selected for the next virulence screening.

[0050] Table 2 Results of the second toxicity comparison

[0051] Strain name Deaths mortality rate Strain name Deaths mortality rate Strain name Deaths mortality rate 1-3 5 50％ 1-20 7 70％ APP-3 5 50％ 1-5 6 60％ 1-21 6 60％ APP-4 2 20％ 1-9 8 80％ 1-22 3 30％ APP-5 3 30％ 1-10 6 60％ 1-25 4 40％ APP-6 2 20％ 1-11 2 20％ 1-26 5 50％ APP-7 1 10％ 1-12 3 30％ 1-27 3 30％ APP-8 5 50％ 1-16 6 60％ APP-2 7 70％ Normal saline 0 0％

[0052] Thirty 40-day-old piglets were randomly divided into 6 groups, with 5 pigs in each group. Five pigs were used as negative controls and injected with normal saline. The other 5 groups were injected with the 5 strains selected above through the trachea, with a challenge dose of 5×10 7 / piglets were observed for 7 consecutive days, and the incidence and mortality of piglets were recorded. The results are shown in Table 3. The lung scores after autopsy were as follows: Figure 1 shown.

[0053] Table 3 Piglet challenge screening results

[0054] Strain name Number of piglets Deaths mortality rate 1-9 5 2 40％ 1-16 5 5 100％ 1-20 5 4 80％ 1-26 5 5 100％ APP-2 5 4 80％ Normal saline 5 0 0％

[0055] Based on the mortality of piglets and the scoring of lung lesions after autopsy, although the mortality rate of strain 1-26 also reached 100%, it did not cause serious lung lesions compared with strain 1-16. Therefore, 1-16 was finally selected as the candidate immune strain and named APP2022116.

[0056] The applicant sent the strain to the China Center for Type Culture Collection for preservation on July 21, 2023, with classification name: Actinobacillus pleuropneumoniae APP2022116, preservation number: CCTCC NO: M20231360, address: Wuhan University, Wuhan, China.

[0057] 2. Identification of the immunogenicity of Actinobacillus pleuropneumoniae APP2022116

[0058] 1) Preparation of inactivated vaccine against Actinobacillus pleuropneumoniae

[0059] Swine Actinobacillus pleuropneumoniae APP2022116 was spread on a TSA plate containing 10% newborn calf serum and 0.01% NAD, cultured in a 37°C constant temperature incubator overnight for 12 hours, washed with PBS and inactivated by adding formaldehyde at a final concentration of 0.4%, placed in a shaker at 37°C and 200 rpm for 48 hours, centrifuged and washed twice, and resuspended in PBS.

[0060] Take the inactivated porcine pleuropneumoniae Actinobacillus APP2022116 and mix it with aluminum hydroxide adjuvant to make each dose of vaccine contain 1×10 bacteria 9 indivual.

[0061] 2) Evaluation of the immune protection effect of inactivated vaccine against porcine Actinobacillus pleuropneumoniae

[0062] Twenty 35-day-old piglets were randomly divided into a control group and a vaccine-immunized group, with 10 piglets in each group. Immunization and challenge were performed according to the experimental scheme in Table 4. A second immunization was performed 28 days after the first immunization, and the immunization method was intramuscular injection in the back of the neck. The immunization dose and route were the same as the first immunization. Before the challenge, the control group and the immunized group were divided into 2 groups, each with 5 piglets, and the APP1 and APP7 strains were used for challenge, respectively (see the immunization process). Figure 2 )

[0063] Table 4 Immune challenge test plan

[0064]

[0065] 14 days after the second immunization, each group was challenged with freshly cultured APP1 and APP7 strains, with the challenge dose of 3×10 7 / Only with 4×10 9 / pig. Observe the clinical symptoms and mortality of pigs within one week, perform autopsy on dead pigs, collect various tissue parts, recover the diseased tissue and fix it with 4% formaldehyde. At the end of the observation, euthanize the pigs that are not dead, and observe the changes in their autopsy. The criteria for judging the onset of the disease: ① Depression; ② Coughing and difficulty breathing; ③ Death; ④ Hemorrhagic fibrinous pleuropneumonia or focal necrosis can be seen in the autopsy of asymptomatic pigs. Those who have any of the above items are judged to be onset. Statistical analysis of the immune protection rate of the vaccine.

[0066] The test results showed that the inactivated vaccine had a cross-protective effect against APP1 and APP7. A total of 5 pigs died in the PBS+adjuvant control group during the observation period, including 3 pigs in the APP1 challenge control group, 2 pigs in the APP7 challenge control group, and 1 pig in the immunization group, which was caused by APP1 challenge. The protection rates of the inactivated vaccine against pigs challenged with APP1 and APP7 strains were 80% and 100%, respectively. The results are shown in Table 5.

[0067] Table 5 Immune protection results of porcine Actinobacillus pleuropneumoniae (APP2022116 strain) inactivated vaccine

[0068] Group Challenge strain Number of cases Deaths mortality rate Number of cases Incidence Immunization group APP1 type 1 1 20％ 1 20％ Control group APP1 type 5 3 60％ 5 100％ Immunization group APP7 0 0 0％ 0 0％ Control group APP7 5 2 40％ 5 100％

[0069] Embodiment 2:

[0070] ApxⅠA and ApxⅡA protein expression and purification:

[0071] 1. Construction and verification of recombinant plasmid:

[0072] ApxⅠA (shown in SEQ ID NO.3, encoding the protein shown in SEQ ID NO.1) and ApxⅡA (shown in SEQ ID NO.4, encoding the protein shown in SEQ ID NO.2) were constructed into pET-28a(+), pET-30a(+), pET-32a(+), pGEX-6P-1, pCold-Ⅰ, pCold-sumo, and pCold-TF vector plasmids, respectively.

[0073] The specific method is as follows:

[0074] By designing primers for ApxⅠA and ApxⅡA genes (Table 6), using the genomic DNA of APP2022116 as a template, PCR was performed according to the procedure in Table 7 to amplify the target gene fragments ApxⅠA and ApxⅡA, respectively. Since PrimerSTAR Max DNA Polymerase has the characteristics of high specificity, high reaction sensitivity, and high amplification efficiency, and its own high annealing efficiency greatly shortens the annealing and extension time, according to the instructions, the PCR of the target gene can use the reaction program of pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 10s; annealing at 52℃ for 10s; extension at 72℃ for 45s; and final extension at 72℃ for 2min, 30 cycles. After the PCR was completed, nucleic acid electrophoresis was performed, and the results showed that the band sizes of ApxⅠA and ApxⅡA amplified were located near the expected positions.

[0075] Table 6 Primer sequences

[0076]

[0077] Table 7 PCR reaction system

[0078] Reaction components Volume (μL) 2×PrimerSTAR Max DNA Polymerase 25 Upstream primer 2 Downstream primer 2 Genome template 1 <![CDATA[ddH2O]]> 20 total 50

[0079] The pET-28a(+), pET-30a(+), pET-32a(+), pGEX-6P-1, pCold-Ⅰ, pCold-sumo, and pCold-TF vectors were double-digested with the amplified ApxⅠA and ApxⅡA, and EcoRI and BamHI were selected for digestion at 37℃ for 4h. After 4h of digestion, the digestion products were treated with Omega Gel Extraction Kit was used for gel recovery, and then the recovered ApxⅠA and ApxⅡA fragments were connected with pET-28a(+), pET-30a(+), pET-32a(+), pGEX-6P-1, pCold-Ⅰ, pCold-sumo, and pCold-TF vectors, respectively, to obtain a total of 14 recombinant plasmids.

[0080] The product was transformed into E.coli DH5α competent cells, and then the bacterial solution was spread on LA plates containing the corresponding antibiotics for resistance screening. The obtained monoclonal clone was inoculated into LB medium containing the corresponding antibiotics, and after shaking and culturing at 37°C for 12 hours, the plasmid was extracted for PCR identification and sent to Qingke Biotechnology Company for sequencing identification.

[0081] These recombinant expression plasmids were identified by PCR and sequencing, and all were consistent with expectations, with no sequence mutations, proving that the genes were successfully inserted into the corresponding vectors.

[0082] 2. Expression and purification of recombinant target protein

[0083] The recombinant plasmid verified by sequencing was transferred into E. coli BL21 (DE3) competent cells, and then the bacteria were spread on LA plates containing the corresponding antibiotics for resistance screening. Single clones of the recombinant strain were picked and inoculated into LB liquid culture medium containing the corresponding antibiotics. 600 When the pH reaches 0.6-0.8, take an appropriate amount of bacterial solution as a blank control (without IPTG), add IPTG with a final concentration of 1mM to the remaining bacterial solution, and induce protein in each recombinant bacterium at 16℃ for 16h. After the induction, collect the bacteria by centrifugation, resuspend and break them, collect the supernatant and precipitate, and perform SDS-PAGE electrophoresis to detect the expression of the target protein.

[0084] After the induction expression conditions were determined, the newly streaked recombinant strain was inoculated and transferred to 1L LB liquid medium for expansion culture, and shaken at 37°C and 200 rpm until the OD 600When the pH value is 0.6-0.8, IPTG with a final concentration of 1 mM is added to induce each recombinant bacterium under its optimal induction conditions. After the induction is completed, the bacteria are collected, crushed under high pressure, centrifuged at low temperature, and the supernatant and the precipitate are separated.

[0085] After optimizing the expression vector and expression conditions, it was found that only the two recombinant proteins of the pCold-TF plasmid could be expressed in a soluble form. The induction expression conditions were 16°C, adding IPTG with a final concentration of 1mM, and inducing expression at 170rpm for 24h.

[0086] The recombinant protein was then purified using conventional Ni resin affinity chromatography and detected by SDS-PAGE. It was found that the protein could not bind to the Ni resin affinity layer regardless of the type of loading buffer or the corresponding solvent concentration.

[0087] The applicant tried to replace the purification and recovery materials and used anion and cation exchange columns for recovery, but found that the target protein still could not bind to the filler.

[0088] Finally, after completing protein induction, the applicant crushed the protein expression supernatant under high pressure, adjusted the pH value of the loading buffer (20mM Tris-Hcl) to 11, and used a pre-packed column containing a large-pore anion exchange filler Rigose-DEAE to load the sample through an AKTA pure25 protein chromatography purification system. The loading flow rate was 1mL / min. During the loading process, the impurities in the supernatant were combined with the anion exchange filler, and the target protein was retained in the loading flow-through. After collecting the flow-through, a 10000MWCO ultrafiltration tube was used to obtain ApxIA and ApxIIA proteins by ultrafiltration concentration. The recombinant protein was subjected to SDS-PAGE detection. The results showed that the sizes of the purified ApxIA and ApxIIA were 163kDa and 155kDa, respectively, which were consistent with expectations ( Figure 2 ).

[0089] Embodiment 3:

[0090] Preparation of inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae and evaluation of its immune effect

[0091] 1. Preparation of inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae

[0092] The APP2022116 bacteria were spread onto TSA plates containing 10% newborn calf serum and 0.01% NAD, cultured in a 37°C constant temperature incubator overnight for 12 h, washed with PBS, and inactivated by adding formaldehyde at a final concentration of 0.4%. The plates were placed in a shaker at 37°C and 200 rpm for 48 h, washed twice by centrifugation, and resuspended in PBS.

[0093] Take the purified proteins ApxIA and ApxIIA and dilute them appropriately according to their concentrations and mix them evenly. Mix the diluted proteins and inactivated porcine Actinobacillus pleuropneumoniae serotype 1 bacteria with CPC adjuvant so that each dose of vaccine contains 250 μg of each protein and 1×10 porcine Actinobacillus pleuropneumoniae serotype 1 bacteria. 9 indivual.

[0094] 2. Evaluation of the immune effect of inactivated subunit combination vaccines

[0095] Take 20 35-day-old piglets and randomly divide them into a control group and a vaccine immunization group, with 10 piglets in each group. Perform immunization according to the experimental scheme in Table 8. A second immunization was performed 28 days after the first immunization, and the immunization method was intramuscular injection in the neck and back. The immunization dose and route were the same as the first immunization. Blood was collected from the anterior vena cava every 7 days before and after immunization, and the serum was packaged and frozen for later use. Before the challenge, the control group and the immunization group were each divided into 2 groups, with 5 piglets in each group, and the challenge strains APP1 and APP7 in Example 1 were used for challenge, respectively (see the immunization process). Figure 2 ).

[0096] Table 8 Immune challenge test plan

[0097]

[0098] 3. Serum specific antibody level detection

[0099] The recombinant proteins ApxⅠA and ApxⅡA were diluted to 500 ng / mL with PBS and coated on ELISA plates, 100 μL per well, overnight at 4°C, and then blocked with 5% skim milk at 37°C for 2 h. The serum before and after immunization (1:500) was used as the primary antibody, and goat anti-swine IgG-HRP (1:5000) was used as the secondary antibody. After sufficient incubation, TMB colorimetric solution was used for color development for 10 min, and the color development was stopped with colorimetric stop solution, and then the OD was read with an enzyme reader. 630 The numerical values ​​of ApxⅠA and ApxⅡA specific antibody levels in the serum of each group were detected and compared.

[0100] The results showed that after booster immunization, the levels of ApxⅠA and ApxⅡA specific antibodies in the serum of mice in each group were significantly increased compared with those before the initial immunization ( Figure 4 ). This showed that the vaccine could stimulate pigs to produce high levels of specific antibodies against the above two antigens.

[0101] 4. Virus challenge test

[0102] 14 days after the second immunization, each group was challenged with freshly cultured APP1 and APP7 strains in Example 1, with the challenge doses of 6×10 7 / Only with 4×10 9 / pig. Observe the clinical symptoms and mortality of pigs within one week, perform autopsy on dead pigs, collect various tissue parts, recover the diseased tissue and fix it with 4% formaldehyde. At the end of the observation, euthanize the pigs that are not dead, and observe the changes in their autopsy. The criteria for judging the onset of the disease: ① Depression; ② Coughing and difficulty breathing; ③ Death; ④ Hemorrhagic fibrinous pleuropneumonia or focal necrosis can be seen in the autopsy of asymptomatic pigs. Those who have any of the above items are judged to be onset. Statistical analysis of the immune protection rate of the vaccine.

[0103] The results showed that a total of 6 deaths occurred in the PBS+adjuvant control group during the observation period, including 4 deaths in the APP1-type challenge control group, 2 deaths in the APP7-type challenge control group, and no deaths in the immunization group ( Figure 5 The protection rate of the inactivated subunit combined vaccine against pigs challenged with APP1 and APP7 strains was 100% (5 / 5), and the results are shown in Table 9. At the same time, by comparing the lung autopsy of the control group and the vaccine group, it was shown that the lungs of the pigs in the control group were severely adhered to the chest cavity, the lungs were severely hemorrhaged, the lungs were enlarged, caseous necrosis was visible in some parts, the tracheal mucosa was hemorrhaged, and there was foam or blood-red foam in the trachea; the lungs of the pigs in the control group were mostly intact, with basically no adhesion between the lungs and the chest cavity, with small lumps visible to the naked eye, and the inner wall of the trachea was smooth and without bleeding ( Figure 6 , 7 ). The results showed that the vaccine had a good immune protection effect on pigs infected with APP1 and APP7 strains.

[0104] Table 9 Immune protection rate of inactivated subunit combined vaccine against different strains after infection

[0105]

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of an inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae in preparing a porcine Actinobacillus pleuropneumoniae vaccine, wherein the porcine Actinobacillus pleuropneumoniae is porcine Actinobacillus pleuropneumoniae serotype 1 and / or serotype 7; The vaccine comprises: rApxⅠ protein shown in SEQ ID NO.1, rApxⅡ protein shown in SEQ ID NO.2 and inactivated Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae ), the deposit number of the porcine actinobacillus pleuropneumoniae is CCTCC NO: M20231360; The preparation method of the rApxⅠ protein or rApxⅡ protein comprises: The polynucleotide encoding the protein shown in SEQ ID NO.1 or SEQ ID NO.2 is connected to the vector pCold-TF to obtain a recombinant vector, which is then introduced into E. coli BL21 (DE3) for induced fermentation, and the corresponding protein is obtained in the fermentation supernatant. The polynucleotide encoding the protein shown in SEQ ID NO.1 is shown in SEQ ID NO.3, and the polynucleotide encoding the protein shown in SEQ ID NO.2 is shown in SEQ ID NO.4; The fermentation induction expression conditions are 16°C, adding IPTG with a final concentration of 1 mM, and inducing expression at 170 rpm for 24 hours; The fermentation supernatant is separated and purified by the following method: The pH value of the loading buffer was adjusted to 11, and the sample was loaded using a prepacked column containing a large-pore anion exchange filler Rigose-DEAE through an AKTA pure25 protein chromatography purification system. The loading flow rate was 1 mL / min. During the loading process, the impurities in the supernatant combined with the anion exchange filler, and the target protein was retained in the loading flowthrough. After collecting the flowthrough, a 10000 MWCO ultrafiltration tube was used to obtain rApxⅠ or rApxⅡ protein by ultrafiltration concentration.

2. Use of an inactivated subunit combined vaccine of porcine Actinobacillus pleuropneumoniae in the preparation of a drug for treating or preventing porcine Actinobacillus pleuropneumoniae infection, wherein the porcine Actinobacillus pleuropneumoniae is porcine Actinobacillus pleuropneumoniae serotype 1 and / or 7; The vaccine comprises: rApxⅠ protein shown in SEQ ID NO.1, rApxⅡ protein shown in SEQ ID NO.2 and inactivated Actinobacillus pleuropneumoniae ( Actinobacillus pleuropneumoniae ), the deposit number of the porcine actinobacillus pleuropneumoniae is CCTCC NO: M20231360; The preparation method of the rApxⅠ protein or rApxⅡ protein comprises: The polynucleotide encoding the protein shown in SEQ ID NO.1 or SEQ ID NO.2 is connected to the vector pCold-TF to obtain a recombinant vector, which is then introduced into E. coli BL21 (DE3) for induced fermentation, and the corresponding protein is obtained in the fermentation supernatant. The polynucleotide encoding the protein shown in SEQ ID NO.1 is shown in SEQ ID NO.3, and the polynucleotide encoding the protein shown in SEQ ID NO.2 is shown in SEQ ID NO.4; The fermentation induction expression conditions are 16°C, adding IPTG with a final concentration of 1 mM, and inducing expression at 170 rpm for 24 hours; The fermentation supernatant is separated and purified by the following method: The pH value of the loading buffer was adjusted to 11, and the sample was loaded using a prepacked column containing a large-pore anion exchange filler Rigose-DEAE through an AKTA pure25 protein chromatography purification system. The loading flow rate was 1 mL / min. During the loading process, the impurities in the supernatant combined with the anion exchange filler, and the target protein was retained in the loading flowthrough. After collecting the flowthrough, a 10000 MWCO ultrafiltration tube was used to obtain rApxⅠ or rApxⅡ protein by ultrafiltration concentration.

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