Bovine type A Pasteurella multocida PM1790 protein and Pm1790 gene and their application

Abstract

The present invention discloses a bovine type A Pasteurella multocida PM1790 protein and Pm1790 gene and their applications, relating to the technical field of biological products. The amino acid sequence of the bovine type A Pasteurella multocida PM1790 protein is shown in SEQ ID NO. 1. The present invention also discloses a bovine type A Pasteurella multocida PM1790 gene encoding the bovine type A Pasteurella multocida PM1790 protein, the nucleotide sequence of which is shown in SEQ ID NO. 2. The present invention also discloses the application of the bovine type A Pasteurella multocida PM1790 protein or Pm1790 gene in the preparation of a Pasteurella multocida vaccine. Vaccines prepared using strains lacking the Pm1790 gene can induce high levels of antibody production and effectively prevent infection by various strains of Pasteurella multocida.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to a bovine type A Pasteurella multocida PM1790 protein and a Pm1790 gene and applications thereof. Background Art

[0002] Pasteurella multocida (P. multocida) is a broadly host-tropic opportunistic pathogen that can infect birds and mammals, causing a variety of diseases such as avian cholera, rabbit plague, porcine atrophic rhinitis, and bovine respiratory disease syndrome. This has posed a serious threat to the global livestock and poultry industry and wildlife. Furthermore, there are reports that P. multocida can infect humans through bites or excrement from pets such as dogs and cats, leading to a variety of diseases such as arthritis, meningitis, peritonitis, and sepsis. With rising living standards and the increasing popularity of pet ownership, the number of deaths caused by P. multocida infection is increasing annually, posing a serious threat to human health. However, since its isolation in the last century, the pathogenic mechanism of P. multocida remains unclear, and commercial vaccines are limited. Therefore, understanding the pathogenic mechanism of this bacterium and developing new prevention and control technologies are of great significance. Summary of the Invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a bovine type A Pasteurella multocida PM1790 protein and Pm1790 gene and their applications. The vaccine prepared using a strain lacking the Pm1790 gene can induce high-level antibody production and effectively prevent infection by various strains of Pasteurella multocida.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: a bovine type A Pasteurella multocida PM1790 protein is provided, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] The present invention also provides a bovine type A Pasteurella multocida Pm1790 gene encoding a bovine type A Pasteurella multocida PM1790 protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] The present invention also provides a use of the bovine type A Pasteurella multocida PM1790 protein or the bovine type A Pasteurella multocida Pm1790 gene in the preparation of a Pasteurella multocida vaccine.

[0007] The present invention also provides an inactivated vaccine against Pasteurella multocida, comprising a bovine type A Pasteurella multocida gene-deficient strain; the bovine type A Pasteurella multocida gene-deficient strain is based on the bovine type A Pasteurella multocida and lacks the bovine type A Pasteurella multocida Pm1790 gene.

[0008] Furthermore, the bovine type A Pasteurella multocida is the PmCQ2 strain.

[0009] The present invention has the following beneficial effects:

[0010] 1. This study confirms that PM1790 is an important virulence factor of PmCQ2. PM1790 protein can induce macrophages to secrete cytokines such as IL-6, IL-12p40, IL-17, TNF-α, IL-1β, and IFN-γ. It also exhibits cytotoxicity and can cause cell apoptosis.

[0011] 2. The present invention successfully deleted the Pm1790 gene from the bovine type A Pasteurella multocida strain PmCQ2 through homologous recombination, significantly reducing the virulence of PmCQ2 and significantly attenuating its pathogenicity. Furthermore, an inactivated vaccine prepared using the Pm1790 gene-deleted strain exhibits strong cross-protective efficacy, effectively preventing infection with various types of Pasteurella multocida. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the gel electrophoresis result of Pm1790 gene amplification;

[0013] Figure 2 This is the result diagram of PM1790 protein purification verification;

[0014] Figure 3 This is the result of ELISA test on PM1790 protein inducing macrophages to secrete cytokines;

[0015] Figure 4 This is the result of qRT-PCR detection of cytokine secretion by macrophages induced by PM1790 protein;

[0016] Figure 5 Figure 2 shows the results of cell morphology observation and cytotoxicity detection of macrophages infected with PM1790 protein.

[0017] Figure 6 Flowchart for the construction of the PmCQ2-Δ1790 gene deletion strain;

[0018] Figure 7 This is a diagram showing the identification results of the gene-deficient strain PmCQ2-Δ1790 and the gene-complemented strain PmCQ2-C1790;

[0019] Figure 8This is a graph showing the antibody titer of the inactivated vaccine against PmB and PmP strains;

[0020] Figure 9 This is a graph showing the antibody titer of the inactivated vaccine against PmCQ2 and PmQ strains;

[0021] Figure 10 This is a graph showing the antibody titer of the inactivated vaccine against the PmF strain;

[0022] Figure 11 This is a graph showing the antibody titer of the inactivated vaccine against the C45-2 strain;

[0023] Figure 12 This is the immune protection rate of the inactivated vaccine against the PmCQ2 strain;

[0024] Figure 13 This is the immune protection rate of the inactivated vaccine against PmB and PmF strains;

[0025] Figure 14 This is a graph showing the immune protection rate of inactivated vaccines against PmP and PmQ strains;

[0026] Figure 15 This is a graph showing the immune protection rate of the inactivated vaccine against the C45-2 strain. DETAILED DESCRIPTION

[0027] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0028] Example 1

[0029] A bovine-derived Pasteurella multocida type A PM1790 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0030] Example 2

[0031] A bovine type A Pasteurella multocida Pm1790 gene encodes a bovine type A Pasteurella multocida PM1790 protein, and its nucleotide sequence is shown in SEQ ID NO.2.

[0032] Experimental Example 1: Pm1790 gene amplification

[0033] The total DNA of bovine type A Pasteurella multocida PmCQ2 bacteria was extracted according to the operating instructions of the bacterial genomic DNA extraction kit of TIANGEN.

[0034] BioXM 2.6 software was used to design specific primers for the Pm1790 gene of the PmCQ2 strain (as shown in Table 1) and synthesized by Shanghai Bioengineering Co., Ltd.

[0035] Table 1 Specific primers for Pm1790 gene

[0036]

[0037] The PCR amplification reaction system using the extracted PmCQ2 bacterial total DNA as a template and PM1790F / R specific primers is shown in the following table. Reaction conditions: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 90 seconds, for a total of 35 cycles; 72°C extension for 10 minutes. The amplified product was detected by 1.0% agarose gel electrophoresis. The results are as follows. Figure 1 As shown in Figure 2, lane M is a DNA marker (DL1500), lane 1 is an amplified fragment of the Pm1790 gene, and lane 2 is a negative control. The results showed that the amplified gene fragment was 1017 bp in length, consistent with the target gene fragment length. Furthermore, the amplified product was recovered using an agarose gel purification kit and sent to Shanghai Bioengineering Co., Ltd. for sequencing to confirm that the amplified product was correct.

[0038] Experimental Example 2 Induced expression and purification of PM1790 protein

[0039] The stored pET-30a(+) bacterial liquid was inoculated on a solid culture medium containing Kan resistance and cultured. The plasmid was extracted according to the operating instructions of the plasmid extraction kit and the concentration was detected. The extracted plasmid was double-digested with Bam HI and Hind III endonucleases, and the digestion product was ligated with the Pm1790 gene.

[0040] The ligation product was transferred into competent E. coli DH5α cells and positively screened on LB solid medium containing Kan resistance. The screened recombinant positive plasmid was then transformed into the E. coli host strain BL21(DE3). After verification, an expression strain containing the Pm1790 gene was obtained and stored at -80°C until use.

[0041] The expression strain containing the recombinant plasmid was activated and cultured in LB liquid medium (containing 0.1% Kan) with shaking until the absorbance reached 0.4-0.6A. Sterile IPTG was added to induce expression, and then the bacteria were collected by centrifugation. An appropriate amount of PBS was added to the bacterial precipitate, ultrasonically disrupted, and the supernatant was collected after centrifugation. His-Ni-NTA column was selected and purified using NPI-20 / 40 / 100 / 250 (imidazole solution). The eluate and washing liquid of each section were collected separately for SDS-PAGE electrophoresis analysis. The protein electrophoresis results were compared and the target protein purification liquid with clear bands and no miscellaneous bands was selected. The electrophoresis results were as follows: Figure 2 As shown, lane 1 is Protein Marker (150 kDa), lane 1 is the uninduced group, lane 2 is the induced group, and lane 3 is the purified group. The results show that the rPM1790 protein was successfully induced and expressed, the band size was consistent with the expectation, and the purification effect was good.

[0042] After desalting and removing endotoxins from the rPM1790 protein, the protein was tested using the ToxinSensor™ Endotoxin Detection System. The final endotoxin content of the purified protein was 0.0132 EU / mL. The maximum working concentration of the protein in this assay was 15 μg / mL, which meets the standard for cell-based assays specified in the instructions. The protein concentration was also measured using a modified Bradford assay. After desalting and removing endotoxins, the protein concentration was 851.92 μg / mL.

[0043] Test Example 3 Cell Test

[0044] (1) Effect of rPM1790 protein on the secretion of inflammatory factors in mouse macrophages

[0045] Healthy C57BL / 6 mice of appropriate age were selected and stimulated by intraperitoneal injection of thioglycolate. The peritoneal cavity was flushed with RPMI 1640 medium, and the peritoneal lavage fluid was extracted to obtain primary mouse macrophages.

[0046] Place a 12-well plate on the clean bench and 5Macrophages were added to cell / well and allowed to adhere to the wall in a 37°C, 5% CO2 incubator for 2-4 hours. The culture medium was removed, washed 2-3 times with PBS, and an equal amount of culture medium was added. At the same time, 5μg, 10μg and 15μg of desalted and endotoxin-free rPM1790 protein were added thereto and incubated with the culture medium. At the same time, in order to exclude the influence of other factors, the following control groups were set up: LPS (1μg / mL), Boiled (10μg / mL Pro, incubated at 100°C for 1h), PK+Pro (50μg / mL+10μg / mL), and UI was a blank culture medium. The cells were cultured under the same culture conditions as above for 24 hours, centrifuged at 5000rpm for 5 minutes, and the supernatant and precipitate were collected respectively. The supernatant was tested by ELISA according to the instructions of the cytokine detection kit, and RNA was extracted from the precipitate for fluorescence quantitative qRT-PCR analysis to detect the expression of inflammatory factors. The results are as follows. Figure 3 and Figure 4 As shown, ** indicates a significant level of P < 0.01, *** indicates a significant level of P < 0.001, and **** indicates a significant level of P < 0.0001.

[0047] Depend on Figure 3 and Figure 4 It can be seen that, without the interference of external factors, rPM1790 protein can induce macrophages to secrete a large amount of cytokines such as IL-6, IL-12p40, IL-17, TNF-α, IL-1β and IFN-γ. Therefore, PM1790 protein may be involved in the immune response of macrophages to PmCQ2 infection.

[0048] (2) Crystal violet staining of infected macrophages and determination of cytotoxicity by LDH method

[0049] After incubating rPM1790 protein with mouse macrophages for 24 hours, crystal violet staining was used to observe cell morphological changes. At the same time, mouse macrophages were treated with 0 μg / mL, 5 μg / mL, 10 μg / mL, and 15 μg / mL of rPM1790 protein, and the culture supernatant was collected after 24 hours to determine the LDH content. The cytotoxicity of rPM1790 protein was quantitatively analyzed. The results are as follows: Figure 5 As shown, *** indicates a significant level of P < 0.001, and **** indicates a significant level of P < 0.0001.

[0050] Depend on Figure 5It can be seen that after 24 hours of co-incubation of rPM1790 protein with macrophages, microscopic observation showed that the cell morphology was significantly wrinkled, the intercellular spaces were widened, and a large number of cells died and floated in the culture medium compared with the control group. Therefore, it is speculated that rPM1790 protein is cytotoxic and can cause different degrees of morphological changes and even death in primary mouse macrophages. The destruction of cell membrane structure caused by cell death will lead to the release of intracellular enzymes including lactate dehydrogenase (LDH). After rPM1790 protein was treated with mouse macrophages, the LDH content in the culture supernatant increased significantly with the increase of rPM1790 protein concentration, indicating that it is clearly cytotoxic.

[0051] Experimental Example 4 Construction of PmCQ2-Δ1790 gene deletion strain

[0052] The construction process of the PmCQ2-Δ1790 gene deletion strain is as follows Figure 6 As shown. Using the extracted PmCQ2 genome as a template, primers were designed to amplify the upper and lower homologous arms of the Pm1790 gene and then connected. pUC19oriKan was extracted from the preserved strain. R The plasmid was then double-digested with BamHI and HindIII endonucleases to obtain a linearized cloning vector, and then Exnase TM II ligase ligated the ligated upper and lower homology arms of the Pm1790 gene to the linearized cloning vector. Finally, the recombinant plasmid was electroporated into PmCQ2 competent cells, and the gene deletion strain PmCQ2-Δ1790 was selected using specific primers.

[0053] At the same time, the full-length gene of Pm1790 was amplified and ligated into the double-digested pUC19oriKan R Plasmid formation pUC19oriKan R -C1790 recombinant plasmid, and then transformed into PmCQ2-Δ1790 competent cells, and the positive bacterial liquid was selected for culture to obtain the stably inherited gene-complemented strain PmCQ2-C1790.

[0054] The gene deletion strain PmCQ2-Δ1790 and the gene complementation strain PmCQ2-C1790 were verified by qRT-PCR and Western Blot for Pm1790 gene expression. Figure 7 As shown in the table, * indicates a significant level of P < 0.05, and **** indicates a significant level of P < 0.0001. The results were consistent with expectations, indicating that the gene deletion strain and gene complementation strain were successfully constructed.

[0055] Test Example 6 Inactivated vaccine

[0056] (1) Preparation of inactivated vaccine: PmCQ2-Δ1790 cultured to the logarithmic growth phase was transferred to 100 mL of Martin liquid medium at a ratio of 1:50 for expansion culture. After dilution and counting, the cells were centrifuged at 10,000 rpm for 15 min in a low-temperature centrifuge. The supernatant was discarded and the cells were resuspended in an appropriate culture medium. Formaldehyde solution was added according to the proportion to a final concentration of 0.15%. The cells were placed in a 37°C constant temperature incubator for 24 h. During this period, the cells were vortexed every 2 h to ensure that all bacteria were inactivated. The inactivated bacterial solution was added to Martin liquid medium at a ratio of 4:1 and the mineral oil adjuvant 15VG was added and mixed to emulsify the cells to prepare a culture medium emulsifier and PmCQ2-Δ1790 inactivated vaccine. The inactivated vaccine was plated to confirm the presence of live bacteria. At the same time, the emulsifier and inactivated vaccine were injected subcutaneously into the backs of two KM mice to observe the reactions of the mice and conduct safety tests. After treatment, no foreign bacteria grew on the vaccine smear, proving that the inactivated vaccine passed the sterilization test; after one week of observation, the mice injected subcutaneously with the vaccine were in good mental state and did not produce any pathological phenomena. It was preliminarily determined that the vaccine was safe.

[0057] (2) Inactivated vaccine cross-protection assay: One hundred female KM mice of appropriate age were randomly divided into 10 groups, with 10 mice in each group. Each group received a primary immunization of culture medium emulsifier and PmCQ2-Δ1790 inactivated vaccine. A secondary booster immunization was performed 14 days after the primary immunization. The immunization dose and challenge strain are shown in Table 2.

[0058] Table 2 Cross-immunization doses and challenge strain types

[0059]

[0060] (3) Inactivated vaccine serum antibody titer detection: One week after the second immunization, the tail vein of the mice was blood collected and the serum was separated. The whole bacterial protein of PmB, PmCQ2, PmF, PmP, PmQ and C45-2 strains was used as antigen, and the antibody titer level of the vaccine against different strains was detected by ELISA. The results are as follows: Figure 8-11 shown.

[0061] Depend on Figure 8-11 It can be seen that after vaccination with the inactivated vaccine, the serum antibody level of mice can reach a high level of more than 1:51200.

[0062] (3) Challenge: One week after the second immunization, mice were challenged with inactivated vaccines to test the cross-protection of the inactivated vaccine. The challenge doses were 10 CFU PmQ (LD 50 ≈1CFU), 2×10 7 CFU PmCQ2(LD 50 =3.3×10 5 CFU), 1.2×10 7 CFU PmB (LD50 =5.0×10 3 CFU)、5×108CFU PmF(LD 50 =1×10 8 CFU)、10CFU PmP(LD 50 ≈1CFU) and 10CFUC45-2(LD 50 ≈1 CFU), and observed every 12 hours after the infection for one week, and recorded the death of mice in detail. Figure 12-15 shown.

[0063] Depend on Figure 12-15 It can be seen that the PmCQ2-Δ1790 inactivated vaccine has different degrees of protection against PmB, PmCQ2, PmF, PmP and C45-2. Among them, it can even reach 100% for PmCQ2, and more than 70% for PmB and PmF, indicating that the inactivated vaccine has good immune protection and can be used as a candidate for commercial vaccine development.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a bovine type A Pasteurella multocida PM1790 protein or a bovine type A Pasteurella multocida PM1790 gene in the preparation of a Pasteurella multocida vaccine; The amino acid sequence of the bovine type A Pasteurella multocida PM1790 protein is shown in SEQ ID NO.1; The bovine type A Pasteurella multocida PM1790 gene encodes the bovine type A Pasteurella multocida PM1790 protein, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. An inactivated vaccine against Pasteurella multocida, characterized in that: It comprises a bovine type A Pasteurella multocida gene-deficient strain; the bovine type A Pasteurella multocida gene-deficient strain is based on the bovine type A Pasteurella multocida and lacks the bovine type A Pasteurella multocida Pm1790 gene described in claim 1.

3. The inactivated vaccine against Pasteurella multocida according to claim 2, wherein The bovine type A Pasteurella multocida is the PmCQ2 strain.

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