Bordetella pertussis engineering strain as well as preparation method and application thereof

Abstract

The invention discloses a bordetella pertussis engineering strain as well as a preparation method and application thereof. Compared with a Bordetella pertussis strain (Bordetella pertussis), the Bordetella pertussis engineering strain disclosed by the invention is capable of expressing an lpxD protein from Pseudomonas jinjiensis (Pseudomonas jinjiensis), and the Bordetella pertussis engineering strain disclosed by the invention can be used for expressing the lpxD protein from Pseudomonas jinjiensis (Pseudomonas jinjiensis). The Bordetella pertussis engineering strain provided by the invention has the effect of obviously increasing the yield of OMV, and can promote the development of Bordetella pertussis OMV vaccines.

Description

Technical Field

[0001] This invention belongs to the field of vaccinology, specifically to Bordetella pertussis (…). Bordetella (B.) pertussis This relates to the prevention or treatment of infections. Specifically, it involves an engineered strain of Bordetella pertussis, its preparation method, and its applications. Background Technology

[0002] Whooping cough, also known as pertussis, is caused by Bordetella pertussis (…). Bordetella (B.) pertussis Pertussis is a highly contagious respiratory disease caused by Bordetella pertussis (also known as Bordetella pertussis). Pertussis is characterized by a violent coughing attack, followed by forceful inhalation and a distinctive whooping cough sound. Bordetella pertussis was first identified as the causative agent of pertussis in 1906. Bordetella pertussis is a Gram-negative bacterium that specifically infects humans. The bacteria are transmitted from person to person through inhaled respiratory droplets. During the 1940s and 1950s, a whole-cell pertussis (wP) vaccine was used against this bacterium, consisting of inactivated Bordetella pertussis combined with diphtheria and tetanus toxoids. Due to some serious complications of the wP vaccine, it was replaced by an acellular (aP) vaccine in the late 1990s. The aP vaccine contains a combination of some of the most important Bordetella pertussis virulence factors, such as pertussis toxin, filamentous hemagglutinin, Bordetella pertussis adhesin, and fimbriae 2 and 3.

[0003] However, pertussis persists in individuals vaccinated with the aP vaccine, and epidemiological data report an increase in pertussis incidence worldwide in recent years, possibly related to the evolution of pertussis strains and the shortened duration of protection provided by the aP vaccine. To prevent the resurgence of pertussis, there is an urgent need to develop a more effective new vaccine.

[0004] In recent years, more and more people have begun to pay attention to the use of outer membrane vesicles (OMVs) as vaccines, such as OMV-based Neisseria meningitidis vaccines. Neisseria meningitidis The vaccine BEXSERO, containing the OMV of the New Zealand strain NZ98 / 254, was approved for marketing by the European EMA and the US FDA in 2013 and 2015, respectively. US9623102B2 uses OMVs of Bordetella pertussis and Bordetella parapertussis to induce a strong immune response, reduce bacterial colonization, and ensure long-term effectiveness.

[0005] OMVs (also known as "outer membrane vesicles") are double-membrane structures, typically spherical, with a diameter ranging from 20-250 nm (sometimes 10-500 nm). They bud off from the outer membrane of Gram-negative bacteria. The OMV membrane contains phospholipids (PL) internally and lipopolysaccharides (LPS) and PL externally, mixed with membrane proteins at different locations, largely reflecting the structure of the bacterial outer membrane from which it buds off. The lumen of an OMV may contain various compounds from the periplasm or cytoplasm, such as proteins, RNA / DNA, and peptidoglycan (PG). However, unlike bacterial cells, OMVs lack the ability to self-replicate. OMV vaccines offer several advantages: First, OMVs can carry most bacterial substances, including key antigens on the bacterial surface, but lack the ability to replicate. Second, OMVs possess natural adjuvant properties, strongly stimulating both innate and adaptive immune responses. Third, OMVs exhibit good stability to high temperatures and certain chemicals. These characteristics demonstrate the strong potential of OMVs as a novel form of bacterial vaccine.

[0006] However, *Bordeza pertussis* cannot secrete high levels of OMV, which greatly limits the development of pertussis OMV vaccines. Reports on increasing *Bordeza pertussis* OMV production are few. For example, Eline F. de Jonge et al. (Curr Res MicrobSci. 2022 Nov 12:3:100172.) found that simultaneous knockout of the *mlaF* and *pldA* genes in *Bordeza pertussis* led to the accumulation of outer membrane phospholipids and increased OMV production. Eline F de Jonge et al. also found that conditional knockout of the *pal* gene could also increase *Bordeza pertussis* OMV production (Res Microbiol. 2022 May-Jun;173(4-5):103937.). Furthermore, knockout of the *mltA* gene (CN 118853525 B) or specific point mutations in the *OmpA* protein (CN116438193A) of *Bordeza pertussis* also have the effect of increasing OMV production. Besides the targets mentioned above, few other novel targets for increasing OMV production have been reported in Bordetella pertussis. Therefore, there is an urgent need to find new targets to accelerate the development of Bordetella pertussis OMV vaccines. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an engineered strain of Bordetella pertussis, its preparation method, and its application.

[0008] Specifically, the present invention solves the above-mentioned technical problems through the following technical solutions: The first aspect of the present invention provides an engineered strain of Bordetella pertussis, wherein the engineered strain of Bordetella pertussis is similar to a Bordetella pertussis strain (…). Bordetella pertussisCompared to ), expressing Jinzhou Pseudomonas ( Pseudomonas jinjuensis lpxD protein from ).

[0009] In some implementations, the Bordetella pertussis strain is BAA 589.

[0010] In some implementations, the lpxD protein is expressed via a promoter.

[0011] In some implementations, the promoter is an endogenous or exogenous promoter capable of initiating gene expression in the BAA 589 strain.

[0012] In some implementations, the promoter is selected from the pompp promoter, pptx promoter, and J23119 promoter.

[0013] In some specific implementations, the promoter is the pompp promoter.

[0014] In some embodiments, the lpxD protein comprises an amino acid sequence as shown in SEQ ID NO: 1, or the gene encoding the lpxD protein comprises a nucleotide sequence as shown in SEQ ID NO: 2.

[0015] In some implementations, the pompp promoter comprises a nucleotide sequence as shown in SEQ ID NO: 3.

[0016] A second aspect of the present invention provides a nucleic acid construct comprising encoding Pseudomonas jinjuans (… Pseudomonas jinjuensis The gene for the lpxD protein.

[0017] In some implementations, the nucleic acid construct further includes a promoter for expressing the gene.

[0018] In some implementations, the promoter is the pompp promoter.

[0019] In some embodiments, the lpxD protein comprises an amino acid sequence as shown in SEQ ID NO: 1, or the gene comprises a nucleotide sequence as shown in SEQ ID NO: 2.

[0020] A third aspect of the present invention provides a method for preparing engineered strains of Bordetella pertussis, the method comprising: To Bordetella pertussis strain ( Bordetella pertussis The nucleic acid construct as described in the second aspect of the present invention is introduced into the present invention.

[0021] A fourth aspect of the present invention provides a method for preparing pertussis outer membrane vesicles, the method comprising culturing an engineered strain of Bordetella pertussis as described in the first aspect of the present invention, and obtaining pertussis outer membrane vesicles from the culture.

[0022] The fifth aspect of the present invention provides pertussis epithelial vesicles prepared as described in the fourth aspect of the present invention.

[0023] The sixth aspect of the present invention provides the use of the nucleic acid construct as described in the second aspect of the present invention in the preparation of engineered strains of Bordetella pertussis.

[0024] The seventh aspect of the present invention provides the use of engineered strains of Bordetella pertussis as described in the first aspect of the present invention in the preparation of pertussis outer membrane vesicles.

[0025] An eighth aspect of the present invention provides a composition comprising at least two of the following: (1) The engineered strain of Bordetella pertussis according to the first aspect of the present invention; (2) The pertussis epithelial vesicles according to the fifth aspect of the present invention; (3) Adjuvants and / or pharmaceutically acceptable carriers.

[0026] The ninth aspect of the invention provides the use of engineered strains of Bordetella pertussis according to the first aspect of the invention, the pertussis outer membrane vesicles according to the fifth aspect, or the compositions according to the eighth aspect in the preparation of medicaments or vaccines for the prevention and / or treatment of Bordetella pertussis infection.

[0027] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0028] The reagents and raw materials used in this invention are all commercially available.

[0029] The positive and progressive effects of this invention are as follows: The strain of Bordetella pertussis engineered strain provided by this invention has a significant effect on increasing OMV production, which will promote the development of Bordetella pertussis OMV vaccine. Attached Figure Description

[0030] Figure 1 Map of gene editing plasmids.

[0031] Figure 2 This is the result of PCR verification of the strain.

[0032] Figure 3 For I2:: PjlpxD Sequencing results of the strain.

[0033] Figure 4The OMV concentration and particle number of the bacterial culture before purification were determined.

[0034] Figure 5 To determine the concentration of purified OMV.

[0035] Figure 6 The number of particles was determined for the purification of OMV.

[0036] Figure 7 The particle size was determined for the purification of OMV. Detailed Implementation

[0037] definition

[0038] OMV: outer membrane vesicles, referring to the outer membrane vesicles secreted by Gram-negative bacteria.

[0039] IPTG: Isopropyl β-D-thiogalactoside, is a commonly used inducer that can induce the expression of target genes as needed.

[0040] NTA: Nanoparticle tracking analysis is a method for detecting the particle size distribution of samples in liquid suspensions by utilizing the properties of light scattering and Brownian motion.

[0041] Pseudomonas jinjuensis The lpxD gene can synthesize short acyl chain LOS. When the lpxD gene in *Botrytis cinerea* is replaced by the expression of PjlpxD, theoretically the acyl chain of the *Botrytis cinerea* LOS will become shorter, thus achieving strain attenuation. Therefore, those skilled in the art generally use... PjlpxD Toxicity reduction 。

[0042] The present invention is further illustrated below by way of examples, but these examples do not limit the invention to the scope of the embodiments described. Experimental methods not specifically described in the following examples were performed according to conventional methods and conditions, or as selected according to the product instructions. For illustrative purposes, the present invention uses the BAA-589 strain purchased from ATCC (American Type Culture Collection).

[0043] Example 1: Strain Construction

[0044] 1.1 Plasmid Construction Information

[0045] from Pseudomonas jinjuensisThe amino acid sequence encoding the LpxD protein was obtained from the bacteria and designated as PjLpxD (SEQ ID NO: 1). The PjLpxD sequence was codon-optimized for expression in Bordetella pertussis, yielding the optimized sequence. PjlpxD The nucleotide sequence (SEQ ID NO: 2) was obtained, and the gene was synthesized from this sequence. The synthesized gene fragment was then obtained via PCR. PjlpxD Fragments. Additionally, the leader promoter region derived from the Outer Membrane Protein P gene, namely the pompp fragment, was obtained from the genome of the starting strain BAA-589 via PCR amplification (primer sequences are shown in Table 1, and the nucleotide sequence of the pompp fragment is shown in SEQ ID NO: 3). The two obtained fragments were inserted into the plasmid backbone via homologous recombination. Further colony PCR and plasmid sequencing were used to ensure the correctness of the target sequence on the plasmid. The plasmid map is shown in... Figure 1 As shown.

[0046] Table 1.

[0047] 1.2 Plasmid transformation and strain validation

[0048] The gene-edited plasmid was introduced into competent *Bordetella pertussis* cells via electroporation. The electroporation parameters were: voltage 1.6-2.2 kV, resistance 200 Ω, capacitance 25 μF, and electroporation time not exceeding 5 ms. After electroporation, SS liquid medium was added, and the cells were incubated at 35-37℃ and 220-240 rpm for recovery. After recovery, the cells were centrifuged, most of the supernatant was discarded, and the resuspended bacterial cells were plated onto BG plates containing 10 μg / ml gentamicin resistance. The plates were then incubated statically at 35-37℃. After single colonies of suitable size grew on the electroporated plates, PCR verification was performed on the single colonies. The verification primers are shown in Table 2 below. Figure 2 The validation results show that the PCR product size of clone 6 is as expected, indicating that... PjlpxD The gene has been successfully integrated into the genome of Bordetella pertussis, and the target strain has been obtained, denoted as I2:: PjlpxD The strain (where I2 is the non-functional region of Bordetella pertussis).

[0049] Table 2.

[0050] Furthermore, regarding I2:: PjlpxD The PCR products of the strain were sequenced to confirm that the edited sequence of the target strain was consistent with the expected sequence. Figure 3 As shown, the sequencing results indicate PjlpxD The gene has been correctly inserted into the target site, and the strain sequence is correct.

[0051] Example 2: OMV Production Measurement

[0052] After obtaining the strain, the target strain I2:: PjlpxD OMV production was determined. Colonies from the agar plate were inoculated into 4 ml of SS liquid medium and cultured at 35°C and 240 rpm in a shaker to obtain the seed culture. After approximately 20 hours of seed culture culture, the seed culture was transferred to a shake flask containing 200 ml of SS medium and cultured at 35°C and 240 rpm in a shaker. After approximately 45 hours of transfer, the OMV concentration in the supernatant was determined. The determination method was as follows: 1 ml of bacterial culture was centrifuged at 10,000 rpm for 5 minutes, the supernatant was collected, filtered through a 0.22 µm filter, and the filtered supernatant was diluted appropriately. The OMV particle concentration was then determined using a NanoSight Pro instrument according to the instruction manual. Figure 4 As shown, I2:: PjlpxD The OMV concentration of the strain was 1.97E11 / ml of bacterial culture, which was significantly higher than the OMV concentration of 3.15E10 / ml of the control strain, indicating that expression... PjlpxD Genes can increase the number of OMV particles secreted by Bordetella pertussis.

[0053] In addition, the fermentation broth was collected for OMV purification. The OMV purification method is as follows: the bacterial broth was centrifuged at 4000 rpm for 60 min at 4℃ to obtain the supernatant, which was then filtered using a 0.22 µm filter. The filtered supernatant (140 ml each) was ultracentrifuged twice, first using a large rotor (ultracentrifugation conditions: 4℃, 33000 rpm for 1.5 h), then using a small rotor (ultracentrifugation conditions: 4℃, 50000 rpm for 1.5 h). After ultracentrifugation, 0.6 ml of buffer was added to resuspend the OMV, and the OMV was then filtered through a 0.22 µm filter for sterilization. The filtered OMV samples were used for subsequent analysis.

[0054] The protein concentration of purified OMV samples was determined using the BCA method. Figure 5 As shown, the OMV protein concentration of the control strain was 0.13 mg / ml OMV, I2:: PjlpxD The OMV protein concentration of the strain was 1.28 mg / ml OMV. Compared with the control strain, I2:: PjlpxD The strain showed an approximately 9.8-fold increase in OMV protein concentration, indicating expression... PjlpxD Genes can increase the production of Bordetella pertussis OMV.

[0055] The number of particles in the purified OMV was determined using a NanoSight Pro instrument. Figure 6As shown, the control strain had an OMV particle count of 1.64 E12 / ml OMV, I2:: PjlpxD The strain had an OMV particle count of 3.36E13 / ml OMV. I2:: PjlpxD The number of OMV particles in the strain was approximately 20-fold higher than that in the control strain, indicating that... PjlpxD Gene expression can increase the OMV secretion of Bordetella pertussis.

[0056] In addition, the particle size and polydispersity index (PDI) of the purified OMV were determined using a Malvern DLS instrument. Figure 7 As shown, I2:: PjlpxD The OMV particle size of the strain was approximately 97 nm, which is within the range of OMV particle size for Bordetella pertussis. Furthermore, the PDI value of the purified OMV was approximately 0.2, indicating good homogeneity of the OMV.

[0057] Sequence information: PjLpxD amino acid sequence (SEQ ID NO: 1) MMTAPTFSLADLAAQLGAELRGDATLVIRGLATLQDAAPDQLSFLANPQYRKFLPGSQAGAVLLTAADADGYAGNALVVANPYLAYASLSHLFDPKPRAAAGIHPTAIVDSSASVDPSASVGPYVVIEAGASIGPGVTLGAHCFIGARSSIGEGGWLAPRVTLYHDVRIGKRVSIQ SGAVLGGEGFGFANEKGVWQKIAQIGGVSIGDDVEIGANTTIDRGALSDTLVGNGVKLDNQIMIAHNVQIGDHTAMAGCCGISGSAKIGSHCMLAGGVGLVGHIEICDNVFVTGMTMVTRSITEPGSYSSGTAMQPAAEWKKSAARIRQLDDMARRLQQLEKRLAAVTSDGDTPSDA codon optimization PjlpxD Gene nucleotide sequence (SEQ ID NO: 2) pompp nucleotide sequence (SEQ ID NO: 3) GATGAACCATGCATACAACCTATTGAATCTTCACAGTTAGCCCGCGCGCGATTCCGGATTAAGGGGACAGGATGTTGCAACTTACCAACAATGGGGCGGGAAACCGGCTTTTTTCTGAGCCTGACCATAGCCAGTCCTGCCGATTTTTGATGCAATAGCGTCAACTTCCCTTCGCGGAGTTAGGGGGGGCGGCAGGCCGGTATGTGCTGCATTGCTGCTCTTGTCACTCAAATCAACGGAGATTTCTTAA Plasmid backbone nucleotide sequence (SEQ ID NO: 4) Upstream nucleotide sequence of the plasmid backbone insertion site (SEQ ID NO: 5) TGGCCGAGGTGCCCATCAGG Downstream nucleotide sequence of the plasmid backbone insertion site (SEQ ID NO: 6) CTCGAGCCAGGCATCAAATA While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An engineered strain of Bordetella pertussis, characterized in that, The engineered strain of Bordetella pertussis and the strain of Bordetella pertussis ( Bordetella pertussis Compared to ), expressing Jinzhou Pseudomonas ( Pseudomonas jinjuensis lpxD protein from ).

2. The engineered strain of Bordetella pertussis as described in claim 1, characterized in that, The Bordetella pertussis strain is BAA 589; and / or, The lpxD protein comprises the amino acid sequence shown in SEQ ID NO: 1, or the gene encoding the lpxD protein comprises the nucleotide sequence shown in SEQ ID NO:

2.

3. A method for preparing engineered strains of Bordetella pertussis, characterized in that, The method includes: To Bordetella pertussis strain ( Bordetella pertussis The code introduced in ) is derived from Pseudomonas jinjuense ( Pseudomonas jinjuensis The gene for the lpxD protein.

4. The method as described in claim 3, characterized in that, The lpxD protein comprises an amino acid sequence as shown in SEQ ID NO: 1, or the gene comprises a nucleotide sequence as shown in SEQ ID NO:

2.

5. A method for preparing pertussis outer membrane vesicles, characterized in that, The method includes culturing the engineered strain of Bordetella pertussis as described in claim 1 or 2, and obtaining pertussis outer membrane vesicles from the culture.

6. Pertussis epithelial vesicles prepared by the method of claim 5.

7. The application of a nucleic acid construct in the preparation of engineered strains of Bordetella pertussis; said nucleic acid construct contains encoding compounds derived from Pseudomonas jinzhouense (… Pseudomonas jinjuensis The gene for the lpxD protein.

8. The application as described in claim 7, characterized in that, The lpxD protein comprises an amino acid sequence as shown in SEQ ID NO: 1, or the gene comprises a nucleotide sequence as shown in SEQ ID NO:

2.

9. The use of the engineered strain of Bordetella pertussis as described in claim 1 or 2 in the preparation of pertussis outer membrane vesicles.

10. A composition comprising at least two of the following: (1) The engineered strain of Bordetella pertussis according to claim 1 or 2; (2) The pertussis outer membrane vesicles according to claim 6; (3) Adjuvants and / or pharmaceutically acceptable carriers.

11. Use of the engineered strain of Bordetella pertussis according to claim 1 or 2, the pertussis outer membrane vesicle according to claim 6, or the composition according to claim 10 in the preparation of a medicament or vaccine for the prevention and / or treatment of Bordetella pertussis infection.

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