Bovine-derived A-type pasteurella multocida PM1790 protein and Pm1790 gene and application thereof

Abstract

The invention discloses a bovine-derived A-type pasteurella multocida PM1790 protein, a Pm1790 gene and application of the bovine-derived A-type pasteurella multocida PM1790 protein and the Pm1790 gene, and relates to the technical field of biological products, and the amino acid sequence of the bovine-derived A-type pasteurella multocida PM1790 protein is as shown in SEQ ID NO. 1. The invention also discloses a bovine-derived A-type pasteurella multocida Pm1790 gene which is used for coding a bovine-derived A-type pasteurella multocida PM1790 protein, and the nucleotide sequence of the bovine-derived A-type pasteurella multocida Pm1790 gene is as shown in SEQ ID NO. 2. The invention also discloses an application of the bovine-derived type A pasteurella multocida PM1790 protein or the Pm1790 gene in preparation of a pasteurella multocida vaccine. The vaccine prepared from the strain lacking the Pm1790 gene can induce the generation of a high-level antibody, and can effectively prevent the infection of pasteurella multocida of various strain types.

Description

Technical Field

[0001] The 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 conditional pathogen with a wide range of host tropism. It can infect birds and mammals and cause various diseases, such as avian cholera, rabbit plague, porcine atrophic rhinitis, bovine respiratory disease syndrome, etc., which has caused serious harm to the global livestock and poultry industry and the growth and reproduction of wild animals. In addition, there are also literature reports that P.multocida can infect humans through the scratches or excrement of pets such as dogs and cats and cause a variety of diseases, such as arthritis, meningitis, peritonitis, sepsis, etc.; with the improvement of living standards and the promotion of the pet-raising wave, the number of deaths caused by infection with P.multocida is gradually increasing every year, which seriously threatens human life and health. However, since the isolation of P.multocida in the last century, its pathogenic mechanism is still unclear and there are few commercial vaccines. Therefore, it is of great significance to explore the pathogenic mechanism of the bacteria and develop new prevention and control technologies. 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 problem is as follows: a bovine type A Pasteurella multocida PM1790 protein is provided, and its amino acid sequence 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, and the nucleotide sequence thereof is shown in SEQ ID NO.2.

[0006] The present invention also provides an application 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 obtained by deleting the bovine type A Pasteurella multocida Pm1790 gene based on the bovine type A Pasteurella multocida.

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

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

[0010] 1. The present invention 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, and is also cytotoxic and can cause cell apoptosis.

[0011] 2. The present invention deletes the Pm1790 gene in the genome of bovine type A Pasteurella multocida PmCQ2 by homologous recombination, successfully reducing the virulence of PmCQ2 and significantly weakening its pathogenicity. In addition, the inactivated vaccine prepared using the Pm1790 gene-deficient strain has a good cross-immune protection effect and can effectively prevent 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 a diagram showing the PM1790 protein purification verification results;

[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 PM1790 protein inducing macrophages to secrete cytokines;

[0016] Figure 5 This is the result of observation of cell morphology and cytotoxicity test of PM1790 protein infected macrophages;

[0017] Figure 6 This is a 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] Fig. 9 This is a graph showing the antibody titer of the inactivated vaccine against PmCQ2 and PmQ strains;

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

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

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

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

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

[0026] Fig.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, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0028] Example 1

[0029] A bovine type A Pasteurella multocida 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 total PCR reaction system with PM1790F / R specific primers was as follows: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 90s, 35 cycles; 72℃ extension for 10min. The amplified product was detected by 1.0% agarose gel electrophoresis. Figure 1 As shown, lane M is DNA Marker (DL1500), lane 1 is the amplified fragment of Pm1790 gene, and lane 2 is the negative control. The results show that the length of the amplified gene fragment is 1017bp, which is consistent with the length of the target gene fragment. Further, the agarose gel purification kit is used for recovery, and the recovered product is sent to Shanghai Bioengineering Co., Ltd. for sequencing to confirm that the amplified product is correct.

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

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

[0040] The ligation product was transferred into the competent E. coli cell DH5α, and positive screening was performed on LB solid medium containing Kan resistance. The screened recombinant positive plasmid was then transferred into the E. coli host strain BL21 (DE3), and after verification, an expression strain containing the Pm1790 gene was obtained and stored at -80°C for future 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. A His-Ni-NTA column was selected, and NPI-20 / 40 / 100 / 250 (imidazole solution) was used for purification. 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 impurities 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 expectations, and the purification effect was good.

[0042] After desalting and removing endotoxin from rPM1790 protein, the ToxinSensorTM endotoxin content detection system was used to detect the endotoxin content of the purified protein. The endotoxin content of the final protein was 0.0132EU / mL. The maximum working concentration of the protein in this test was 15μg / mL, which met the standards of the cell test in the instructions. At the same time, the Bradford modified method was used to detect the protein concentration. After desalting and removing endotoxin, the protein concentration was 851.92μg / 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 set aside 5×10 5Macrophages were added to cell / well and allowed to adhere to the wall for 2-4 hours in a 37°C, 5% CO2 incubator. 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 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, and the cytotoxicity of rPM1790 protein was quantitatively analyzed. The results are as follows: Figure 5 As shown, *** indicates a significant level P < 0.001, **** indicates a significant level P < 0.0001.

[0050] Depend on Figure 5It can be seen that after rPM1790 protein was co-incubated with macrophages for 24 hours, compared with the control group, microscopic observation showed that the cell morphology was significantly wrinkled, the intercellular gap was widened, and a large number of cells died and floated in the culture medium. Therefore, it is speculated that rPM1790 protein has cytotoxicity and can cause different degrees of morphological changes and even death of 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 used to treat mouse macrophages, the content of LDH in the culture supernatant increased significantly with the increase of rPM1790 protein concentration, indicating that it is obviously 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 strains. 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 connected upper and lower homologous 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 by specific primers.

[0053] At the same time, the full-length gene of Pm1790 was amplified and ligated to the double-digested pUC19oriKan R Plasmid formation pUC19oriKan R -C1790 recombinant plasmid, and then transferred into PmCQ2-Δ1790 competent cells, and the positive bacterial liquid was selected for culture to obtain the stable genetic gene complementation 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. The results are shown in Figure 7 As shown, * 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 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 appropriate culture medium was added to resuspend the cells. Formaldehyde solution was added according to the proportion to a final concentration of 0.15%, and 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 the Martin liquid medium at a ratio of 4:1 and the mineral oil adjuvant 15VG was added and mixed and emulsified to prepare a culture medium emulsifier and PmCQ2-Δ1790 inactivated vaccine. The inactivated vaccine was plated to determine the presence of live bacteria. At the same time, the emulsifier and the inactivated vaccine were injected subcutaneously into the backs of 2 KM mice, and the reactions of the mice were observed for safety testing. After the treatment, no foreign bacteria grew on the vaccine smear plate, proving that the inactivated vaccine passed the sterilization test; after one week of observation, the mice injected with the vaccine subcutaneously were in good mental state and did not produce any pathological phenomena, which preliminarily determined that the vaccine was safe.

[0057] (2) Determination of cross-protection of inactivated vaccines: 100 female KM mice of appropriate age were randomly divided into 10 groups, with 10 mice in each group. They were injected with culture medium emulsifier and PmCQ2-Δ1790 inactivated vaccine as primary immunization, and a secondary booster 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) Serum antibody titer test of inactivated vaccine: 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 tested 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 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 challenge 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 which 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 modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A bovine type A Pasteurella multocida PM1790 protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A bovine type A Pasteurella multocida Pm1790 gene, characterized in that: The nucleotide sequence encoding the bovine type A Pasteurella multocida PM1790 protein of claim 1 is shown in SEQ ID NO.

2.

3. Use of the bovine type A Pasteurella multocida PM1790 protein according to claim 1 or the bovine type A Pasteurella multocida Pm1790 gene according to claim 2 in the preparation of a Pasteurella multocida vaccine.

4. 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 2.

5. The inactivated vaccine against Pasteurella multocida according to claim 4, characterized in that The bovine type A Pasteurella multocida is strain PmCQ2.

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