A modified Bordetella pertussis strain and its application

Abstract

The present invention discloses a modified Bordetella pertussis strain and its application. The modified Bordetella pertussis strain, in the Bordetella pertussis strain BAA-589, does not express the BP2992 protein or expresses an inactive BP2992 protein, does not express the pldA protein or expresses an inactive pldA protein, and does not express the mlaF protein or expresses an inactive mlaF protein. When treated with ultrasound, the Bordetella pertussis strain exhibits a significantly higher foaming phenotype and significantly increased OMV secretion compared to wild-type Bordetella pertussis BAA-589, laying a good technical foundation for the development of a pertussis OMV vaccine.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a modified Bordetella pertussis strain and application thereof. Background Art

[0002] There are many ways to increase the production of bacterial outer membrane vesicles (OMVs), which can be divided into two categories: increasing the yield from the strain and increasing the yield from the OMV production process.

[0003] From the strain perspective, the production can be increased by editing genes related to the OMV secretion process. There are three commonly used strategies. The first strategy is to weaken the binding between the outer membrane and the peptidoglycan layer, making the outer membrane loose and easy to form vesicles for secretion. Outer membrane lipoproteins are the key bridge for the binding of the outer membrane and peptidoglycan. Reducing the content of outer membrane lipoproteins is an important means to reduce the binding between the outer membrane and the peptidoglycan layer. The second strategy is to increase the phospholipid content on the outer membrane to increase the curvature of the outer membrane. The third strategy is to increase the internal pressure of the periplasmic space to promote OMV secretion.

[0004] From the perspective of OMV production technology, the secretion of OMVs can be increased through various induction methods, but compared with OMVs obtained through natural secretion, each induction method has its own shortcomings; detergents can also be used to induce OMV secretion, but the formed OMVs will lose LPS with adjuvant effect and lipoproteins that may be immunogenic; reagents such as EDTA can also be used to induce OMV secretion, but this may reduce the stability of the OMV membrane; heating can also be used to induce OMV secretion, but the heating method may change the phospholipid composition of the OMV membrane; ultrasound can also be used to induce OMV secretion, but the amount of OMV secretion is often unsatisfactory.

[0005] Wild-type strains of B. pertussis naturally secrete very low levels of OMVs, severely limiting their research and industrial production. Currently, reports on increasing OMV production are limited, and the reported yield increases have been suboptimal. For example, Eline F. deJonge et al., at the strain level, knocked out the phospholipid-associated proteins pldA and malF in Bordetella pertussis and found that they could increase the naturally secreted production of OMVs to a certain extent. The double knockout of both pldA and malF genes resulted in an approximately fourfold increase compared to wild-type strains (Current Research in Microbial Sciences, Volume 3, 2022, 100172, ISSN 2666-5174). D Hozbor et al. focused on the OMV production process and found that the use of ultrasound-induced process can also increase the yield of pertussis OMV within a certain limit (Hozbor, D., Rodriguez, M., Fernández, J. et al. Release of Outer Membrane Vesicles from Bordetella pertussis. Curr Microbiol 38,273–278 (1999).).

[0006] In summary, in order to develop a pertussis OMV vaccine, it is urgent to develop a method to increase the production of pertussis OMV. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a modified Bordetella pertussis strain and application thereof.

[0008] Specifically, the present invention provides a modified Bordetella pertussis strain, which does not express BP2992 protein or expresses inactive BP2992 protein compared to Bordetella pertussis strain BAA-589.

[0009] In some embodiments, the Bordetella pertussis strain comprises a mutation in the BP2992 gene, which results in the non-expression of BP2992 protein or the expression of an inactive BP2992 protein.

[0010] In some specific embodiments, the BP2992 gene mutation includes a deletion of the BP2992 gene.

[0011] In some specific embodiments, the BP2992 gene encodes the amino acid sequence shown in SEQ ID NO: 7 or its nucleotide sequence is shown in SEQ ID NO: 6.

[0012] In some embodiments, the Bordetella pertussis strain does not express the pldA protein and / or the mlaF protein; or

[0013] Express inactive pldA protein and / or mlaF protein.

[0014] In some embodiments, the Bordetella pertussis strain is Bordetella pertussis strain BAA-589, which does not express BP2992 protein or expresses inactive BP2992 protein, does not express pldA protein or expresses inactive pldA protein, and does not express mlaF protein or expresses inactive mlaF protein.

[0015] In some embodiments, the Bordetella pertussis strain comprises mutations in the BP2992, pldA, and mlaF genes; or, the Bordetella pertussis strain comprises mutations in the BP2992 and pldA genes; or, the Bordetella pertussis strain comprises mutations in the BP2992 and mlaF genes.

[0016] In some specific embodiments, the pldA gene mutation comprises a deletion of the pldA gene; and / or the mlaF gene mutation comprises a deletion of the mlaF gene.

[0017] In some specific embodiments, the pldA gene encodes the amino acid sequence shown in SEQ ID NO: 11 or the nucleotide sequence shown in SEQ ID NO: 10; and / or the mlaF gene encodes the amino acid sequence shown in SEQ ID NO: 15 or the nucleotide sequence shown in SEQ ID NO: 14.

[0018] In some specific embodiments, the Bordetella pertussis comprises deletion mutations of the BP2992 gene, the pldA gene, and the mlaF gene.

[0019] The present invention also provides a method for producing a modified Bordetella pertussis strain, the method comprising:

[0020] A mutation is introduced into the BP2992 gene of a Bordetella pertussis strain, thereby obtaining a modified Bordetella pertussis strain with a BP2992 gene mutation, thereby resulting in no expression of BP2992 protein or expression of inactive BP2992 protein.

[0021] In some specific embodiments, the method comprises:

[0022] Introducing mutations into the BP2992 gene, the pldA gene, and the mlaF gene in the Bordetella pertussis strain to obtain a modified Bordetella pertussis strain in which the BP2992 gene, the pldA gene, and the mlaF gene are all mutated; or,

[0023] Introducing mutations into the BP2992 gene and the pldA gene in the Bordetella pertussis strain to obtain a modified Bordetella pertussis strain in which both the BP2992 gene and the pldA gene are mutated; or,

[0024] introducing mutations into the BP2992 gene and the mlaF gene in the Bordetella pertussis strain to obtain a modified Bordetella pertussis strain in which both the BP2992 gene and the mlaF gene are mutated;

[0025] Wherein, the Bordetella pertussis strain is Bordetella pertussis strain BAA-589.

[0026] The present invention also provides a method for preparing pertussis outer membrane vesicles, the method comprising using ultrasound to treat the Bordetella pertussis strain described in any one of the present disclosures.

[0027] In some embodiments, the ultrasonic treatment does not lyse the bacteria of the Bordetella pertussis strain, but promotes the secretion of pertussis outer membrane vesicles of the bacteria.

[0028] In some embodiments, the method comprises the following steps:

[0029] a) culturing the Bordetella pertussis strain according to any one of the present disclosure;

[0030] b) using ultrasonic treatment of the strain obtained in step a) to produce pertussis outer membrane vesicles.

[0031] In some embodiments, for a bacterial solution with a concentration of 20-40 OD / 10 ml, the ultrasonic treatment conditions are selected from one or more of the following:

[0032] (1) Ultrasonic power is 65-90W;

[0033] (2) The total ultrasound time is 3-7 minutes;

[0034] (3) The pulse mode is intermittent pulse, the on-cycle time is 3-5s, and the off-cycle time is 5-10s.

[0035] In some embodiments, the conditions of the ultrasonic treatment are selected from one or more of the following:

[0036] (1) Ultrasonic power is 75%-85%;

[0037] (2) The total ultrasound time is 3-7 minutes;

[0038] (3) The pulse mode is intermittent pulse, the on-cycle time is 3-5s, and the off-cycle time is 5-10s.

[0039] In some specific embodiments, the conditions of the ultrasonic treatment are:

[0040] (1) Ultrasonic power is 84.5W;

[0041] (2) The total ultrasound time was 5 min;

[0042] (3) The pulse mode is intermittent pulse, the on-cycle time is 5s, and the off-cycle time is 5s.

[0043] The present invention also provides a use of the Bordetella pertussis strain described in any one of the present disclosures.

[0044] In some specific embodiments, the application is application in preparing pertussis outer membrane vesicles.

[0045] The present invention also provides pertussis outer membrane vesicles prepared by the method described in any one of the present disclosures.

[0046] The present invention also provides a composition comprising at least two of the following:

[0047] (1) The Bordetella pertussis strain according to any one of the present disclosure;

[0048] (2) The pertussis outer membrane vesicles according to the present disclosure;

[0049] (3) Adjuvant and / or pharmaceutically acceptable carrier.

[0050] The present invention also provides use of the Bordetella pertussis strain according to the present disclosure, the pertussis outer membrane vesicles according to the present disclosure, or the composition according to the present disclosure in the preparation of a drug or vaccine for preventing and / or treating Bordetella pertussis infection.

[0051] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0052] The reagents and raw materials used in the present invention are commercially available.

[0053] The positive progress effect of the present invention is:

[0054] The present invention provides a new Bordetella pertussis strain that does not express the pldA, mlaF and BP2992 genes. When treated with ultrasound, the strain has a significantly high-foaming phenotype compared with the wild-type Bordetella pertussis BAA-589, and the secretion of OMVs is significantly improved, laying a good technical foundation for the development of pertussis OMV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the map of the homologous recombination backbone plasmid.

[0056] Figure 2 This is the alignment of the sequencing results of the BAA-589ΔBP2992 strain.

[0057] Figure 3 This is the alignment of the sequencing results of the BAA-589ΔBP2992ΔpldA strain.

[0058] Figure 4 Alignment of sequencing results of BAA-589ΔBP2992ΔpldAΔmlaF knockout strain.

[0059] Figure 5 Comparison of OD concentrations of the BAA-589ΔBP2992ΔpldAΔmlaF strain and the wild-type strain BAA-589 after 24 hours of culture.

[0060] Figure 6 Comparison of protein concentrations of OMVs between the BAA-589ΔBP2992ΔpldAΔmlaF strain and the wild-type strain BAA-589.

[0061] Figure 7 Comparison of sOMV particle concentrations between the BAA-589ΔBP2992ΔpldAΔmlaF strain and the wild-type strain BAA-589.

[0062] Figure 8 This is the particle size distribution diagram of sOMV of BAA-589ΔBP2992ΔpldAΔmlaF strain and wild-type strain BAA-589.

[0063] Figure 9 Comparison of OD concentrations of the BAA-589ΔpldAΔmlaF strain and the wild-type strain BAA-589 after 24 hours of culture.

[0064] Figure 10 Comparison of protein concentrations of OMVs between the BAA-589ΔpldAΔmlaF strain and the wild-type strain BAA-589.

[0065] Figure 11Comparison of sOMV particle concentrations between the BAA-589ΔpldAΔmlaF strain and the wild-type strain BAA-589.

[0066] Figure 12 This is the particle size distribution diagram of sOMV of BAA-589ΔpldAΔmlaF strain and wild-type strain BAA-589. DETAILED DESCRIPTION

[0067] To better understand the present invention, some terms are first defined. Other definitions are listed throughout the detailed description.

[0068] The terms "non-expressed protein" or "inactive protein" refer to decreased protein expression / non-expression or decreased / inactive protein activity caused by pathways such as "gene mutation" or "gene silencing." "Gene silencing" refers to the use of molecular biological techniques to inhibit or block the expression of specific genes in a strain, preventing the production of functional proteins or nucleic acid products. This approach does not involve changes in gene structure and is primarily achieved through epigenetic regulation or RNA interference.

[0069] The term "BP2992 gene" refers to a gene in Bordetella pertussis that encodes the BP2992 protein. The function of this protein is currently unknown to the scientific community, but the present invention has discovered that one of its functions is to affect the secretion of outer membrane vesicles. Therefore, in the present disclosure, expressing "inactive BP2992 protein" can increase the secretion of outer membrane vesicles of Bordetella pertussis under ultrasonic conditions.

[0070] The term "pldA gene" encodes the pldA protein, which is a phospholipase whose phospholipase activity can degrade phospholipids on the outer membrane and release fatty acids.

[0071] The term "mlaF gene" encodes the ATPase-mlaF protein that provides energy for the Mla system and is one of the most conserved proteins in the Mla system.

[0072] The term "ultrasound" refers to the use of the physical effects of high-frequency mechanical vibrations of ultrasound to act on the target. In the present invention, it refers to the use of an ultrasonic cell disruptor to generate ultrasound to act on bacteria, controlling parameters such as ultrasound time, power, and working / intermittent cycles to promote the secretion of outer membrane vesicles by bacteria.

[0073] The present invention is further illustrated by examples below, but the invention is not limited to these examples. Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions, or according to commercial specifications. To illustrate the technical solutions and effects of the present invention, the present invention used the BAA-589 strain purchased from the ATCC (American Type Culture Collection).

[0074] Example 1: Method for modifying Bordetella pertussis genes by homologous recombination

[0075] 1.1 Construction of exogenous DNA donor plasmid

[0076] The exogenous backbone plasmid contains ori-ColE1 / pMB1 / pBR322 / pUC, the transfer elements OriT and traJ, the gentamicin resistance gene (Gen), and the galk anti-screening gene. The sequence of the exogenous backbone plasmid is shown in SEQ ID NO: 1 (the structure of the backbone plasmid is shown in Figure 1 ), wherein the gentamicin resistance gene sequence is shown in SEQ ID NO: 2, and the galk anti-screening gene sequence is shown in SEQ ID NO: 3. The upstream and downstream homology arms of the target gene integration site are inserted between the upstream site-up (sequence shown in SEQ ID NO: 4) and downstream site-down (sequence shown in SEQ ID NO: 5) of the insertion site of the exogenous backbone plasmid to obtain an exogenous DNA donor plasmid.

[0077] 1.2 Electroporation of donor plasmid

[0078] Activate the glycerol bacteria on BG plates (Bordet-Gengo solid plates) and culture at 37°C until colonies grow. Inoculate the colonies into SS liquid culture (Stainer-Scholte liquid medium) to expand the culture.

[0079] Prepare the strain for electroporation by washing multiple times with 10% glycerol buffer at approximately 4°C. Electroporation was performed using an electroporator (BTX, ECM630). Electroporation parameters were set as follows: 1.6-2.2 kV, 200 Ω, 25 μF, and electroporation time no longer than 5 ms. Immediately after electroporation, add SS liquid medium for recovery. Transfer the system to a sterile EP tube and incubate at 35-37°C at 220-240 rpm on a shaker for recovery. After recovery, centrifuge and discard the supernatant. Add SS liquid medium, resuspend the cells, and plate onto BG solid plates containing 10 μg / mL gentamicin (BG-G). Incubate the plates at 35-37°C. A single colony is considered a potential primary recombinant strain (1HR).

[0080] 1.3 Verification of primary recombinant strains

[0081] Use a sterile pipette tip to randomly pick single clones grown on multiple BG-G screening plates and streak them on a new BG-G plate. Dip the remaining bacteria on the pipette tip into 50 μL sterile water as a PCR verification template. Use the upstream homology arm integration-specific verification primers and / or downstream integration-specific verification primers for verification, and select the clone with the correct recombination.

[0082] 1.4 Counter-screening culture and secondary recombinant strain verification

[0083] Select clones that have correctly undergone primary recombination and streak them onto BG plates containing 2% DOG (2-Deoxy-D-galactose). Incubate the plates at 37°C. A single colony that grows is a potential secondary recombinant strain (2HR). Use a sterile pipette tip to randomly pick single colonies grown on multiple BG-2% DOG screening plates and streak them onto new BG plates. Dip the remaining bacteria on the pipette tip into 50 μL of sterile water as a template for PCR verification. Verify with genomic primers outside the genomic region to select clones that have correctly undergone secondary recombination.

[0084] Genomic external primers refer to primers that use the upstream sequence of the upstream homology arm as the forward primer (excluding the upstream homology arm itself) and the downstream sequence of the downstream homology arm as the reverse primer (excluding the downstream homology arm itself).

[0085] Example 2: Knockout of BP2992, pldA, and mlaF, followed by ultrasonic treatment, increased OMV production by several dozen times compared to BAA-589

[0086] A pldA, mlaF, and BP2992 triple knockout strain was constructed, and it was found that the OMV production was dozens of times higher than that of BAA-589 when treated with ultrasound.

[0087] The BP2992 gene in the pertussis BAA-589 strain was knocked out using the homologous recombination method of Example 1. Specifically, a plasmid carrying the upstream homologous arm and downstream homologous arm of the BP2992 gene was electroporated into the pertussis BAA-589 strain. After primary and secondary homologous recombination, sequencing confirmed that BP2992 had been successfully knocked out (sequencing results are shown in FIG. Figure 2 strain BAA-589ΔBP2992.

[0088] The nucleotide sequence of BP2992 is shown in SEQ ID NO: 6, the amino acid sequence of BP2992 protein is shown in SEQ ID NO: 7, the upstream homology arm sequence used in the gene editing process using the homologous recombination method is shown in SEQ ID NO: 8, and the downstream homology arm sequence is shown in SEQ ID NO: 9.

[0089] The pldA gene was knocked out in the strain BAA-589ΔBP2992 using the homologous recombination method of Example 1. Specifically, a plasmid carrying the upstream homologous arm and the downstream homologous arm of the pldA gene was electroporated into the pertussis BAA-589ΔBP2992 strain. After primary and secondary homologous recombination, sequencing confirmed that pldA had been successfully knocked out (sequencing results are shown in FIG. Figure 3 As shown), strain BAA-589ΔBP2992ΔpldA was obtained.

[0090] The nucleotide sequence of the pldA gene is shown in SEQ ID NO: 10, the amino acid sequence of the pldA protein is shown in SEQ ID NO: 11, the upstream homology arm sequence used in the gene editing process using the homologous recombination method is shown in SEQ ID NO: 12, and the downstream homology arm sequence is shown in SEQ ID NO: 13.

[0091] The homologous recombination method of Example 1 was used to knock out the mlaF gene in the strain BAA-589ΔBP2992ΔpldA. Specifically, a plasmid carrying the upstream and downstream homologous arms of the mlaF gene was electroporated into the pertussis BAA-589ΔBP2992ΔpldA strain. After primary and secondary homologous recombination, sequencing confirmed that mlaF had been successfully knocked out (sequencing results are shown in FIG. Figure 4 As shown), strain BAA-589ΔBP2992ΔpldAΔmlaF was obtained.

[0092] The nucleotide sequence of the mlaF gene is shown in SEQ ID NO: 14, the amino acid sequence of the mlaF protein is shown in SEQ ID NO: 15, the upstream homology arm sequence used in the gene editing process using the homologous recombination method is shown in SEQ ID NO: 16, and the downstream homology arm sequence is shown in SEQ ID NO: 17.

[0093] After the strain was successfully constructed, the OMV production of the BAA-589ΔBP2992ΔpldAΔmlaF strain under ultrasonic conditions was further evaluated.

[0094] The BAA-589 and BAA-589ΔBP2992ΔpldAΔmlaF strains activated on the BG plate were inoculated into shake flasks containing 50 ml of SS medium and cultured overnight at 35°C and 220 rpm. Measure the OD600 of the seed solution and take an appropriate amount of the seed solution to re-inoculate into a shake flask containing 200 ml of SS medium, controlling the initial OD600 to 0.4. The test tubes were placed on a shaker at 35°C and 220 rpm for about 24 hours and then the samples were collected. The OD600 of the fermentation broth was measured and the results were as follows: Figure 5 As shown, the triple knockout strain BAA-589ΔBP2992ΔpldAΔmlaF grew slower than the wild-type strain BAA-589. A 200 ml fermentation sample was centrifuged at 4000 rpm for 1 h, and the supernatant and precipitate were collected separately.

[0095] The supernatant of 200 ml of fermentation broth was used to prepare a naturally secreted OMV sample. The supernatant was filtered through a 0.22 μm filter membrane, and 140 ml of the filtrate was concentrated by centrifugation in an ultracentrifuge (WX+ULTRA, Thermo) at 100,000 g for 2 h. After ultracentrifugation, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended in 1 ml of buffer and further filtered through a 0.22 μm filter membrane to obtain the OMV sample, designated nOMV (natural OMV).

[0096] The pellet from 200 ml of fermentation broth was used to prepare ultrasound-induced OMV samples. The pellet was resuspended in 20 ml of buffer (50 mM Tris, 150 mM NaCl, pH 7.4) and dispersed evenly by pipetting. 10 ml of the sample was then removed for subsequent experiments. The bacterial sample was inactivated by heating in a 56°C water bath for 30 min. The inactivated sample was sonicated using a VCX130 ultrasonic cell disruptor (SONICS) at 84.5 W power, 5 s on, 5 s off, and 5 min. (The inventors have found that for bacterial broths with concentrations between 20 and 40 OD / 10 ml, sonication parameters ranging from 65 to 90 W power, 3 to 5 s on, 5 to 10 s off, and 3 to 7 min can promote OMV secretion without disrupting the bacteria.) After sonication, the cells were centrifuged at 4000 rpm for 1 h, and the supernatant was filtered through a 0.22 μm filter. Next, the supernatant was centrifuged using an ultracentrifuge (WX+ULTRA, Thermo) at 30,000 g for 30 minutes to remove large cell debris. 7.56 ml of supernatant was collected from the BAA-589 sample, and 6.93 ml of supernatant was collected from the BAA-589ΔBP2992ΔpldAΔmlaF sample. After ultracentrifugation, the supernatant was removed and ultracentrifuged again at 100,000 g for 2 hours. After ultracentrifugation, the supernatant was discarded and the pellet was collected. The pellet was resuspended in 1 ml of buffer and filtered through a 0.22 μm filter to obtain the OMV sample, designated as sOMV (spontaneous OMV).

[0097] The protein concentration of OMVs was measured using the BCA method, and the results were as follows: Figure 6 As shown in the figure, characterization results show that under the above culture conditions, the BAA-589ΔBP2992ΔpldAΔmlaF strain naturally secreted four times more OMVs (nOMVs) than the wild-type strain (1.2 vs. 0.3). Ultrasonication significantly increased OMV production in all strains. The wild-type strain increased OMV production by fourfold (1.2 vs. 0.3), meaning sOMVs were four times greater than nOMVs. The BAA-589ΔBP2992ΔpldAΔmlaF strain also increased OMV production by 16fold (19.11 vs. 1.2), meaning sOMVs were 16 times greater than nOMVs. This result indicates that under ultrasonication, the BAA-589ΔBP2992ΔpldAΔmlaF strain exhibited a greater fold increase in OMV production than the wild-type strain compared to natural secretion. The sOMVs produced by the BAA-589ΔBP2992ΔpldAΔmlaF strain were 16 times greater than those produced by the wild-type strain (19.11 vs. 1.2). The results showed that under ultrasonic conditions, the OMV production of the triple-knockout strain BAA-589ΔBP2992ΔpldAΔmlaF increased significantly.

[0098] The sOMV particle concentration was measured using a nanoparticle tracking analyzer (Malvern, NanoSight Pro). Figure 7 The characterization results showed that under ultrasonic conditions, the sOMV production of the BAA-589ΔBP2992ΔpldAΔmlaF strain was 23 times that of the wild-type strain.

[0099] The sOMV particle size was measured using a nanometer particle size analyzer (Malvern, Zetasizer Pro). Figure 8 As shown in the figure, both samples had small dispersion, indicating that the sOMV purified by ultrasonic method had higher quality.

[0100] The characterization results showed that under ultrasonic conditions, the OMVs produced by the BAA-589ΔBP2992ΔpldAΔmlaF strain were dozens of times that of the wild type.

[0101] Example 3: Knockout of pldA and mlaF and ultrasonic treatment increased OMV production 3-6 times compared to BAA-589

[0102] Using the homologous recombination method of Example 1 and similar treatments as in Example 2, the strain BAA-589ΔpldA was obtained, and the double knockout strain 589ΔpldAΔmlaF was further obtained.

[0103] After the strain was successfully constructed, the growth of the BAA-589ΔpldAΔmlaF strain and the production of OMVs under natural secretion and ultrasound conditions were further evaluated. According to the culture and detection methods in Example 2, Figure 9 As shown, the growth of BAA-589ΔpldAΔmlaF was similar to that of BAA-589ΔBP2992ΔpldAΔmlaF, but not as good as that of the wild-type strain BAA-589.

[0104] The protein concentration of OMVs was measured using the BCA method, and the results were as follows: Figure 10As shown, under the above culture conditions, the BAA-589ΔpldAΔmlaF strain naturally secreted five times more OMVs (nOMVs) than the wild-type strain (1.4 vs. 0.3). Ultrasonication significantly increased OMV production in both strains. The wild-type strain increased OMV production fourfold (1.2 vs. 0.3), with sOMVs four times as high as nOMVs. The BAA-589ΔpldAΔmlaF strain also increased OMV production threefold (3.84 vs. 1.4), with sOMVs three times as high as nOMVs. These results indicate that under ultrasonication, the BAA-589ΔpldAΔmlaF strain produced a similar fold increase in OMV production compared to natural secretion. The sOMVs produced by the BAA-589ΔpldAΔmlaF strain were three times as high as those produced by the wild-type strain (3.84 vs. 1.2). This result suggests that under ultrasonication, the double-knockout strain BAA-589ΔpldAΔmlaF exhibited a similar OMV production trend to that of the wild-type strain. Therefore, whether by ultrasound or natural secretion, the OMVs produced by the BAA-589ΔpldAΔmlaF strain were 3-5 times that of the wild type.

[0105] The sOMV particle concentration was measured using a nanoparticle tracking analyzer (Malvern, NanoSight Pro). Figure 11 The characterization results showed that under ultrasonic conditions, the sOMV production of the BAA-589ΔpldAΔmlaF strain was 6 times that of the wild-type strain.

[0106] The sOMV particle size was measured using a nanometer particle size analyzer (Malvern, Zetasizer Pro). Figure 12 As shown in the figure, both samples had low dispersion, which again indicated that the sOMVs purified by ultrasound were of higher quality.

[0107] The results show that during natural secretion, the nOMV production of strains BAA-589ΔpldAΔmlaF and BAA-589ΔBP2992ΔpldAΔmlaF was 5-4 times that of the wild type. During sonication, the sOMV production of both strains increased, with the increase in strain BAA-589ΔBP2992ΔpldAΔmlaF being particularly significant, reaching tens of times that of the wild type.

Claims

1. A modified Bordetella pertussis strain, characterized in that The Bordetella pertussis strain is a Bordetella pertussis strain ( Bordetella pertussis ) In BAA-589, BP2992 protein, pldA protein and mlaF protein are not expressed.

2. The Bordetella pertussis strain according to claim 1, wherein In the Bordetella pertussis strain, the BP2992 gene, the pldA gene and the mlaF gene are knocked out.

3. The Bordetella pertussis strain according to claim 2, wherein The BP2992 gene encodes the amino acid sequence shown in SEQ ID NO: 7 or its nucleotide sequence is shown in SEQ ID NO:

6.

4. The Bordetella pertussis strain according to claim 3, wherein The pldA gene encodes the amino acid sequence shown in SEQ ID NO: 11 or its nucleotide sequence is shown in SEQ ID NO: 10; and / or the mlaF gene encodes the amino acid sequence shown in SEQ ID NO: 15 or its nucleotide sequence is shown in SEQ ID NO:

14.

5. A method for producing a modified Bordetella pertussis strain, the method comprising: introducing mutations into the BP2992 gene, the pldA gene, and the mlaF gene in the Bordetella pertussis strain to obtain a modified Bordetella pertussis strain in which the BP2992 gene, the pldA gene, and the mlaF gene are all knocked out; Wherein, the Bordetella pertussis strain is a Bordetella pertussis strain ( Bordetella pertussis )BAA-589.

6. A method for preparing pertussis outer membrane vesicles, characterized in that: The method comprises treating the Bordetella pertussis strain according to any one of claims 1 to 4 using ultrasound.

7. The method according to claim 6, wherein The ultrasonic treatment does not lyse the bacteria of the Bordetella pertussis strain, but promotes the secretion of pertussis outer membrane vesicles of the bacteria.

8. The method according to claim 6 or 7, wherein: The method comprises the following steps: a) cultivating the Bordetella pertussis strain according to any one of claims 1 to 4; b) using ultrasonic treatment of the strain obtained in step a) to produce pertussis outer membrane vesicles.

9. Use of the Bordetella pertussis strain according to any one of claims 1 to 4 in preparing pertussis outer membrane vesicles.

10. A composition comprising at least two of the following: (1) The Bordetella pertussis strain according to any one of claims 1 to 4; (2) Adjuvant and / or pharmaceutically acceptable carrier.

11. Use of the Bordetella pertussis strain according to any one of claims 1 to 4 or the composition according to claim 10 in the preparation of a medicament or vaccine for preventing and / or treating Bordetella pertussis infection.

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