A recombinant expression vector and engineering bacteria of pertussis antigen and application thereof

Abstract

The application provides a pertussis antigen recombinant expression vector and engineering bacteria and application thereof. The pertussis antigen recombinant expression vector comprises a resistance screening gene, an upstream recombinant nucleic acid fragment of a pertussis exotoxin gene and a downstream recombinant nucleic acid fragment, wherein the upstream and downstream recombinant nucleic acid fragments can respectively perform homologous recombination with the upstream and downstream of the pertussis exotoxin gene. The application takes the pertussis bacillus as a host, constructs an engineering bacteria ΔPT strain for producing FHA and PRN by knocking out the PT gene, simplifies the production process of the FHA and PRN antigens, maintains the high purity and immunogenicity of the FHA and PRN antigens, eliminates the interference of the PT protein in the production of the FHA and PRN antigens, avoids the PT protein detoxification process, and eliminates the risk of possible PT protein toxicity reversal.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention relates to a recombinant expression vector for pertussis antigen and an engineered bacterium, as well as their applications. Background Technology

[0002] Pertussis (whooping cough) is an acute respiratory infectious disease caused by Bordetella pertussis. Bordetella pertussis produces various toxins, such as pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), heat-stable endotoxin (ET), heat-labile toxin (HLT), and tracheocytotoxin (TCT). Among these, pertussis toxin, filamentous hemagglutinin, and pertactin are the active ingredients in pertussis vaccines and DPT combined vaccines. Currently, most acellular pertussis vaccine production processes use wild-type Bordetella pertussis strains to ferment the antigen components FHA, PT, and PRN. The production of the stock solution employs a PT / FHA co-purification method. The purified FHA stock solution often contains varying amounts of PT toxin, requiring a series of chemical reagents, such as formaldehyde, to detoxify the antigen FHA with PT. However, detoxified PT is prone to toxicity reversal, and the detoxification process reduces the immunogenicity and purity of FHA. Imported pertussis vaccines, such as the Sanofi Pasteur vaccine, utilize advanced column chromatography to extract and detoxify antigens such as PT, PRN, and FHA separately, and then mix these antigenic components in a specific ratio to formulate the vaccine. This process ensures batch-to-batch consistency of vaccine quality, and the antigen purity is 90-95%, making it suitable for developing DTacP-based combination vaccines. However, this process is complex, and the purification steps are cumbersome.

[0003] Current methods for preparing pertussis antigen all suffer from cumbersome separation and purification processes, which not only affects the stability of vaccine production but also hinders the development of DPT vaccines. Therefore, there is an urgent need to develop strains and vaccines suitable for simplified pertussis antigen separation and purification processes. Summary of the Invention

[0004] To address the above problems, the purpose of this invention is to provide a recombinant expression vector for pertussis antigen, an engineered bacterial strain, and their applications. This invention uses *Bordetella pertussis* as a host and constructs an engineered ΔPT strain for the production of FHA and PRN by knocking out the PT gene. This simplifies the production process of FHA and PRN antigens, maintains high purity and immunogenicity of FHA and PRN antigens, eliminates interference from PT protein during FHA and PRN antigen production, avoids PT protein detoxification processes, and eliminates the risk of potential PT protein toxicity reversal.

[0005] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0006] In a first aspect, the present invention provides a pertussis antigen recombinant expression vector comprising an resistance selection gene, an upstream recombinant nucleic acid fragment of a pertussis exotoxin gene, and a downstream recombinant nucleic acid fragment, wherein the upstream and downstream recombinant nucleic acid fragments of the pertussis exotoxin gene are capable of homologous recombination with the upstream and downstream of the pertussis exotoxin gene, respectively. The pertussis exotoxin gene is shown in SEQ ID NO. 1.

[0007] According to some embodiments of the present invention, the starting vector for the expression vector is pET32a, pET28a, pUC57 or TA, preferably TA.

[0008] According to some embodiments of the present invention, the nucleotide sequence of the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene comprises or is a nucleic acid fragment consisting of consecutive bases from position m to 986 in the nucleotide sequence shown in SEQ ID NO.2, where m is a natural number ≤ 205; preferably, the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene is a nucleic acid fragment consisting of consecutive bases from position 205 to 986 in the nucleoside sequence shown in SEQ ID NO.2.

[0009] According to some embodiments of the present invention, the nucleotide sequence of the downstream recombinant nucleic acid fragment of the pertussis exotoxin gene comprises or is a nucleic acid fragment composed of consecutive bases from position 1 to position n in the nucleotide sequence shown in SEQ ID NO.3, wherein n is a natural number ≥870-1090; preferably, the downstream recombinant nucleic acid fragment of the pertussis exotoxin gene is a nucleic acid fragment composed of consecutive bases from position 1 to position 870 in the nucleoside sequence shown in SEQ ID NO.3.

[0010] According to some embodiments of the present invention, the nucleotide sequence of the forward primer used to amplify the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene includes SEQ ID NO.8 or as shown in SEQ ID NO.8.

[0011] According to some embodiments of the present invention, the nucleotide sequence of the reverse primer used to amplify the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene includes SEQ ID NO.9 or as shown in SEQ ID NO.9.

[0012] According to some embodiments of the present invention, the nucleotide sequence of the forward primer used to amplify the resistance selection gene includes SEQ ID NO.10 or as shown in SEQ ID NO.10.

[0013] According to some embodiments of the present invention, the nucleotide sequence of the reverse primer used to amplify the resistance selection gene includes SEQ ID NO.11 or is shown as SEQ ID NO.11.

[0014] According to some embodiments of the present invention, the nucleotide sequence of the forward primer used to amplify the downstream recombinant nucleic acid fragment of the pertussis exotoxin gene includes SEQ ID NO.12 or is as shown in SEQ ID NO.12.

[0015] According to some embodiments of the present invention, the nucleotide sequence of the reverse primer used to amplify the recombinant nucleic acid fragment downstream of the pertussis exotoxin gene includes SEQ ID NO.13 or is as shown in SEQ ID NO.13.

[0016] According to some embodiments of the present invention, the resistance selection gene is selected from one or more of the following: kanamycin resistance selection gene, tetracycline resistance selection gene, ampicillin resistance selection gene, and chloramphenicol resistance selection gene, preferably a kanamycin resistance selection gene.

[0017] Preferably, the nucleotide sequence of the kanamycin resistance selection gene is shown in SEQ ID NO.4; the nucleotide sequence of the tetracycline resistance selection gene is shown in SEQ ID NO.5; the nucleotide sequence of the ampicillin resistance selection gene is shown in SEQ ID NO.6; and the nucleotide sequence of the chloramphenicol resistance selection gene is shown in SEQ ID NO.7.

[0018] In a second aspect, the present invention provides a recombinant pertussis antigen expression engineered bacterium comprising the recombinant expression vector described in the first aspect of the present invention.

[0019] Thirdly, the present invention provides a method for preparing a recombinant pertussis antigen-expressing engineered bacterium according to the second aspect of the present invention, which includes transforming wild-type Bordetella pertussis competent cells using the recombinant expression vector according to the first aspect of the present invention.

[0020] Preferably, the wild-type Bordetella pertussis is the wild-type Bordetella pertussis strain ATCC-BAA-589.

[0021] Preferably, the conversion is performed by electroconversion; more preferably, the electroconversion includes linearizing the recombinant expression vector and then electroconverting wild-type Bordetella pertussis competent cells at 2200-2800V and 2-8ms.

[0022] According to some embodiments of the present invention, the preparation method further includes screening transformed wild-type Bordetella pertussis competent cells using the resistance selection gene.

[0023] Fourthly, the present invention provides the use of the pertussis antigen recombinant expression vector according to the first aspect of the present invention or the pertussis antigen recombinant expression engineered bacteria according to the second aspect of the present invention in the preparation of pertussis antigen.

[0024] Preferably, the pertussis antigen is filamentous hemagglutinin and / or pertussis adhesivein.

[0025] Fifthly, the present invention provides a method for preparing pertussis antigen, which includes fermentation using the pertussis antigen recombinant expression engineered bacteria described in the second aspect of the present invention.

[0026] According to some embodiments of the present invention, the fermentation includes the following steps:

[0027] (1) The recombinant pertussis antigen-expressing engineered bacteria according to the second aspect of the present invention are inoculated into a petri dish containing Pougon's blood agar medium and cultured at 35-40°C for 40-72 hours;

[0028] (2) Inoculate the bacterial cells obtained in step (1) into MSS medium and incubate at 35-40℃ for 12-36 hours;

[0029] (3) Inoculate the bacterial culture obtained in step (2) into a fermenter containing MSS medium and incubate at 35-40°C for 68-70 hours.

[0030] In a sixth aspect, the present invention provides a pertussis antigen, which is prepared by the preparation method described in the fifth aspect of the present invention.

[0031] Preferably, the pertussis antigen is filamentous hemagglutinin and / or pertussis adhesivein.

[0032] The present invention has at least the following beneficial effects:

[0033] 1. This invention uses Bordetella pertussis as a host and constructs an engineered ΔPT strain for the production of FHA and PRN by knocking out the PT gene. This simplifies the production process of FHA and PRN antigens, maintains the high purity and immunogenicity of FHA and PRN antigens, eliminates the interference of PT protein during the production of FHA and PRN antigens, avoids the PT protein detoxification process, and eliminates the risk of possible PT protein toxicity reversal.

[0034] 2. The engineered ΔPT strain constructed in this invention, combined with the engineered OEPRN-PT strain used for the production of PT antigen, achieves effective separation and purification of pertussis PT antigen, FHA antigen, and PRN antigen without interference between them, greatly simplifying the production and purification process and reducing production costs.

[0035] 3. Compared with the wild-type strain, the fermentation cycle of the engineered strain ΔPT constructed in this invention is significantly shortened, with the fermentation time reduced by more than 15%. Moreover, the growth rate of the ΔPT strain at the end of fermentation is significantly higher than that of the wild-type strain, and it also has higher FHA and PRN protein yields compared to the wild-type strain. Attached Figure Description

[0036] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0037] Figure 1 A schematic diagram of plasmid construction for the recombinant expression vector TA-PTup-Kan-PTdown;

[0038] Figure 2 The PCR electrophoresis results of the recombinant expression vector TA-PTup-Kan-PTdown are shown; where M: Marker; lanes 1-5: parallel samples carrying the recombinant expression vector TA-PTup-Kan-PTdown;

[0039] Figure 3 The results of agarose gel electrophoresis of the linearized recombinant expression vector TA-PTup-Kan-PTdown are shown; where M: Marker; plasmid: TA / Blunt-Zero Cloning Kit.

[0040] Figure 4 Electrophoresis diagrams showing the upstream and downstream insertions in the recombinant expression vector identified by PCR validation; where M: Marker; upper lanes (up) 1-18: upstream insertion site confirmation; lower lanes (down) 1-18: downstream insertion site confirmation;

[0041] Figure 5 Electrophoresis image for identifying PT gene knockout by PCR verification; where M: Marker;

[0042] Figure 6 The results of SDS-PAGE analysis of purified FHA antigen protein are shown; where M: Marker;

[0043] Figure 7 The results of the CHO agglutination experiment of the purified FHA antigen protein are shown below; where A: FHA protein stock solution obtained by fermentation of wild-type strain; B: negative control (PBS solution); C: parallel sample 1 of 100 μg / ml FHA protein stock solution obtained by fermentation of ΔPT strain; D: parallel sample 2 of 100 μg / ml FHA protein stock solution obtained by fermentation of ΔPT strain.

[0044] Figure 8The results of SDS-PAGE analysis of the purified PRN antigen protein are shown; where M: Marker;

[0045] Figure 9 The results of the CHO agglutination experiment of the purified PRN antigen protein are shown below; where A: PRN protein stock solution obtained by fermentation of wild-type strain; B: negative control (PBS solution); C: parallel sample 1 of 100 μg / ml PRN protein stock solution obtained by fermentation of ΔPT strain; D: parallel sample 2 of 100 μg / ml PRN protein stock solution obtained by fermentation of ΔPT strain.

[0046] Figure 10 The results of SDS-PAGE analysis of the purified PT antigen protein;

[0047] Figure 11 The results are obtained from the HPLC analysis of the purified PT antigen protein.

[0048] Figure 12 The results of the CHO agglutination experiment of purified PT antigen protein are shown; where A: purified PT protein stock solution, 1000 pg / ml PTx; B: negative control (PBS solution);

[0049] Figure 13 The results of the CHO agglutination experiment of the detoxified PT protein toxin stock solution are shown; where A: detoxified PT protein toxin stock solution, 100 μg / ml; B: negative control (PBS solution). Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0051] 1. Experimental Materials

[0052] MSS medium: The following ingredients were prepared: 2.5 g / L sodium chloride, 0.02 g / L anhydrous calcium chloride, 0.5 g / L dipotassium hydrogen phosphate, 0.2 g / L potassium chloride, 0.1 g / L magnesium chloride (hexahydrate), 6.1 g / L Tris-HCl, 10.7 g / L monosodium glutamate (monohydrate), 10 g / L hydrolyzed casein (ACID), 0.24 g / L L-proline, and 1 g / L 2,6-O-dimethyl-β-cyclodextrin. The pH was adjusted to 7.45 ± 0.10 with concentrated hydrochloric acid.

[0053] Bacchus agar powder: purchased from Qingdao Haibo Biotechnology Co., Ltd., product number HB8483-1.

[0054] Burgh's blood agar medium: Place 4.4g of Burgh's agar medium powder into an Erlenmeyer flask, add 79ml of ultrapure water and 1ml of glycerol, mix well, seal the flask, and sterilize at 121℃ for 15min. After sterilization, place the medium at room temperature until it cools to approximately 40-45℃, then add 20% by weight of fresh sheep blood to the flask inside a biosafety cabinet.

[0055] Wild-type pertussis strain ATCC-BAA-589: purchased from Beina Biotechnology, trade number: 70009724.

[0056] SBFI enzyme: purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B300241-0300.

[0057] pmeI enzyme: purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B300256-0250.

[0058] Plasmid TA / Blunt-Zero Cloning Kit: Purchased from Novizan Biosciences Co., Ltd., product number C601-01.

[0059] Amplification primer M13primer Mix: purchased from Novizan Biosciences Co., Ltd.

[0060] 2. Detection Method

[0061] For the method of detecting complete adsorption, please refer to Section 3.1.2 "Adsorption Rate Test" of the section on adsorption of acellular pertussis-diphtheria-tetanus combined vaccine in the 2020 edition of the Chinese Pharmacopoeia.

[0062] Example 1: Construction of engineered bacteria for recombinant expression of pertussis antigen

[0063] I. Constructing the electrotransfer fragment PTup-Kan-PTdown

[0064] 1. Construct a fragment containing a homologous arm replacing the PT gene. The specific steps are as follows:

[0065] (1) Using Phanta Max Super-Fidelity DNA Polymerase (Novaza, P505), primers delate All-s3-CZF and delate All-s3-R1, the upstream arm (PTup) was amplified using the genome of the pertussis strain as a template.

[0066] (2) Using delate All-kan-F2 and delate All-s3-CZR as primers and the genome of the pertussis strain as a template, the downstream arm (PTdown) was amplified;

[0067] (3) Using delate All-kan-F1 and fha-kan-R2 as primers and pUC57 plasmid as template, the Kan fragment was amplified;

[0068] (4) Using delate All-s3-CZF and delate All-s3-CZR as primers, and the product obtained from the above steps as a template, the vector fragment PTup-Kan-PTdown was obtained by overlap PCR splicing, with a fragment size of 2585bp.

[0069] 2. Recombinant vector TA-PTup-Kan-PTdown

[0070] according to Figure 1 The schematic diagram shown illustrates the construction of the recombinant vector TA-PTup-Kan-PTdown. The specific steps are as follows:

[0071] The target fragment PTup-Kan-PTdown was recovered by 1% agarose gel electrophoresis. Then, the target fragment was ligated to the cloning vector TA / Blunt-Zero Cloning Kit using recombinase (ClonExpress Ultra One Step Cloning Kit, purchased from Novizan, batch number: C115-01). The ligation was then performed into *E. coli* DH5α competent cells (purchased from Nanjing Novizan Biotechnology Co., Ltd., trade number: C502-02), and colony PCR was used for detection. The amplification primers were M13 primer mix. Results are shown below. Figure 2 This indicates that PTup-Kan-PTdown was successfully ligated to the cloning vector. Next, the positive clones identified by colony PCR were sent to Shanghai Sangon Biotech for sequencing. Finally, clones whose nucleotide sequences completely match the target gene sequence were stored at -80°C in 20% wt% glycerol.

[0072] Table 1. Fragments and primers required for constructing the recombinant vector TA-PTup-Kan-PTdown.

[0073]

[0074] 3. Preparation of engineered bacteria for recombinant expression of pertussis antigen

[0075] (1) Bacterial culture: Activate the glycerol bacterium of wild-type cough strain ATCC-BAA-589. Take an appropriate amount of the glycerol bacterium of wild-type cough strain ATCC-BAA-589 and spread it on a plate of Pogostemon blood agar containing 20% ​​sheep blood. Incubate at 37℃ for 48 h. Remove the plate and scrape off the bacterial growth on the surface using a sampling stick. Inoculate the plate into MSS medium and incubate at 37℃ for 24 h. Measure its OD. 600, making its OD 600 It falls between 1 and 3.

[0076] (2) Preparation of competent cells: Collect the bacterial cells from the above steps, centrifuge at 4℃ for 15 min, resuspend the bacterial cells in sterile ultrapure water pre-cooled at 4℃, wash the bacterial cells twice, centrifuge at 4℃ for 15 min, collect the bacterial cells, resuspend the bacterial cells in 1 ml of 10% sterile glycerol to obtain wild-type strain ATCC-BAA-589 competent cells, and store them in a -70℃ freezer.

[0077] (3) Preparation of transformation fragments: The upstream and downstream sides of the recombinant vector TA-PTup-Kan-PTdown obtained in the above steps were linearized using sbfI restriction enzyme. The target fragment was detected and recovered by 1% agarose gel electrophoresis. The results are shown in the figure. Figure 3 This indicates that the cloning vector was successfully constructed.

[0078] (4) Electroporation and homologous recombination: 2 μg of linear fragment DNA was transferred into the wild-type strain ATCC-BAA-589 competent cells prepared in the above steps at 2500V and 5ms. After liquid culture for 24h, it was spread on solid medium containing kanamycin and cultured for 3-5 days. Single clones were picked for verification.

[0079] II. Identification of Recombinant Engineered Pertussis Bacteria

[0080] 1. Experimental Procedure

[0081] Pick a single clone prepared in step 3 above and place it into a 1.5 ml centrifuge tube containing 10 μl of pertussis liquid medium (i.e., MSS medium). Take 2 μl as a template for PCR verification and streak the single clone at the same time. The verification primers are shown in Table 2.

[0082] 2. Experimental Results

[0083] The verification results show that both the upstream and downstream insertion sites are correct. Figure 4 ), and the PT gene was identified as being knocked out ( Figure 5 The pertussis antigen recombinant expression engineered bacterium ΔPT strain was successfully constructed by successfully knocking out the gene expressing PT toxin protein through gene recombination. This engineered bacterium no longer expresses PT toxin protein.

[0084] Table 2 Primers used for validation of recombinant engineered pertussis bacteria

[0085]

[0086]

[0087] Example 2: Growth and fermentation culture of recombinant pertussis antigen-expressing engineered bacteria

[0088] 100 μL each of the engineered ΔPT strain and the wild-type pertussis strain (WT) verified in Example 1 were inoculated into two Petri dishes containing Boehringer's blood agar and cultured at 37°C for approximately 48 h. Bacterial growth was scraped from the Petri dishes and inoculated into 1 ml of MSS medium to prepare a bacterial suspension. An appropriate amount of the bacterial suspension was inoculated into 100 ml (including replenishment solution) of MSS medium to achieve an initial OD600 of approximately 0.05. The suspension was then cultured on a shaker at 37°C for 16.5 h, and the OD600 value was measured. The results showed that the ΔPT strain reached its peak growth rate (fermentation endpoint) at 44.5 h, while the wild-type strain reached its peak growth rate (fermentation endpoint) at 52.5 h, indicating that the overall fermentation cycle of the ΔPT strain was shortened by more than 15% compared to the wild-type strain. Furthermore, the OD600 values ​​showed that the ΔPT strain had an OD600 value of 6.35 at the fermentation endpoint, while the wild-type strain had an OD600 value of 4.89. Therefore, the growth rate of the ΔPT strain was significantly higher than that of the wild-type strain at the fermentation endpoint. Simultaneously, the concentrations of FHA and PRN antigen proteins produced by the wild-type and ΔPT strains were measured at their respective fermentation endpoints, and the results are shown in Table 4. The results indicate that, compared to the wild-type strain, the ΔPT strain had higher concentrations of both FHA and PRN antigens at the fermentation endpoint, suggesting that the ΔPT strain exhibited higher expression levels of the target proteins at harvest.

[0089] Table 3 Growth rate of ΔPT strain

[0090] Sampling time / h <![CDATA[ΔPT(OD 600 )]]> <![CDATA[WT(OD 600 )]]> 0 0.05 0.05 16.5 0.44 1.01 20.5 0.71 1.34 24.5 1.16 1.97 40.5 4.39 3.63 44.5 6.35 4.07 48.5 5.83 4.67 52.5 4.96 4.89 68.5 4.78 4.57

[0091] Table 4. Expression levels of FHA and PRN proteins at the fermentation endpoint.

[0092]

[0093]

[0094] Example 3: Purification of FHA protein obtained from engineered strain ΔPT

[0095] 1. Experimental Materials

[0096] PB buffer: 7.1 g / L NaH2PO4·2H2O + 1.42 g / L Na2HPO4·12H2O;

[0097] Buffer A-FHA: 50mM PB + 2M urea, pH 6.0;

[0098] Buffer B-FHA: Buffer A-FHA + 50% (g / ml) ammonium sulfate (AMS);

[0099] Buffer C-FHA: 50mM PB + 2M urea + 0.5% Triton X-100, pH 6.0;

[0100] Buffer D-FHA: 50mM PB + 2M urea + 0.15M NaCl, pH 6.0;

[0101] Buffer solution E-FHA: 50mM PB + 2M urea + 0.30M NaCl, pH 6.0.

[0102] 2. Experimental Procedure

[0103] The supernatants of the fermentation cultures of the ΔPT strain and the wild-type strain obtained in Example 2 were used to separate and purify FHA using the following steps:

[0104] (1) After concentrating the supernatant of the fermentation culture, the medium was replaced with 5 times the volume of buffer A-FHA to obtain the ultrafiltration concentrate. 50% (g / ml) buffer B-FHA was slowly added to the ultrafiltration concentrate until the final concentration of ammonium sulfate was 30% (g / ml). After stirring slowly for 1 hour, the mixture was centrifuged at 15000g and 4℃ for 30 minutes. The centrifuged liquid was collected and named the reconstitution supernatant.

[0105] (2) Take the reconstituted supernatant and load it onto a Capto SPimRes chromatography column equilibrated with buffer A-FHA. After loading, wash with buffer C-FHA for 3-5 CV to remove endotoxins. After equilibration with buffer A-FHA for 3 CV again, wash host protein impurities with buffer D-FHA and finally elute with buffer E-FHA to obtain purified FHA protein stock solution. Then perform SDS-PAGE detection and CHO agglutination experiment.

[0106] SDS-PAGE display ( Figure 6 The FHA antigen protein purified by fermentation of strain ΔPT showed a band purity greater than 95% compared to the FHA protein standard, with no interference from PT protein bands, indicating that the presence of PT protein had been eliminated from the purified FHA protein stock solution. CHO agglutination experiments showed ( Figure 7 The purified FHA protein stock solution, compared to the FHA protein stock solution obtained by fermentation with the wild-type strain, showed no cell aggregation at a concentration of 100 μg / ml. These results indicate that the FHA stock solution obtained from the ΔPT strain is free of PT toxin, avoiding the need for subsequent PT detoxification, simplifying the production process, and maintaining high FHA purity and immunogenicity to a certain extent.

[0107] Table 5. SDS-PAGE results of purified FHA protein.

[0108] Sample number Sample Name purity / % Molecular weight / kDa 1 Standard products 95 220 2 Purified FHA antigen stock solution 95.8 220

[0109] Example 4: Purification of PRN protein obtained from engineered strain ΔPT

[0110] 1. Experimental Materials

[0111] Buffer A-PRN: 10mM Tris-HCl + 0.15M NaCl, pH 8.0;

[0112] Buffer B-PRN: 50% AMS (g / ml) + Buffer A;

[0113] Buffer C-PRN: 25mM Tris-HCl + 0.035M NaCl, pH 8.8;

[0114] Buffer D-PRN: 50mM Tris-HCl, pH 8.5;

[0115] Buffer E-PRN: 50 mmol / L Tris-HCl + 0.5% Triton X-100;

[0116] Buffer F-PRN: 50mM Tris-HCl + 50mM NaCl, pH 8.5;

[0117] Buffer G-PRN: 50mM Tris-HCl + 120mM NaCl, pH 8.5.

[0118] 2. Experimental Procedure

[0119] The fermentation cultures of the ΔPT strain and wild-type strain obtained in Example 2 were used to isolate and purify PRN using the following steps:

[0120] (1) The fermentation culture cells were resuspended in buffer A-PRN at a ratio of 1:30 (g / ml) and incubated at 60℃ for 1 h. Then, the mixture was centrifuged at 15000g and 4℃ for 30 min, and the supernatant was collected to obtain the extract.

[0121] (2) Slowly add buffer B-PRN to the extract, stir slowly for 1.5 h, centrifuge at 15000 g for 30 min, and collect the precipitate. Resuspend thoroughly with buffer C-PRN (at 2 ml buffer C-PRN per gram of bacterial cells), reconstitute at 4 °C for 1 h, and then filter through a 0.22-0.45 μm filter membrane to obtain the reconstituted supernatant.

[0122] (3) The reconstituted supernatant was concentrated by ultrafiltration through a 10 kDa membrane and the buffer was changed. The concentrated sample was then subjected to QHP column chromatography, eluted with buffer D-PRN and buffer E-PRN, and equilibrated with buffer D-PRN after elution. Impurities were then washed with buffer F-PRN, followed by elution with buffer G-PRN. The eluent was replaced with 9 g / L NaCl solution and sterilized by filtration in a biosafety cabinet. The obtained filtrate was the purified PRN protein stock solution and stored at 2-8℃. The purified PRN protein stock solution was subjected to SDS-PAGE analysis and CHO agglutination assay.

[0123] SDS-PAGE display ( Figure 8 The PRN protein purified by fermentation of strain ΔPT showed an SDS-PAGE purity of over 95% compared to the PRN protein standard, with no PT protein band interference, indicating that the presence of PT protein had been eliminated from the purified PRN protein stock solution. CHO agglutination experiments showed ( Figure 9 The PRN protein stock solution purified by fermentation of the ΔPT strain showed no cell aggregation at a concentration of 100 μg / ml compared to the PRN protein stock solution obtained by fermentation of the wild strain, indicating that there was no PT toxin in the purified PRN protein solution and no detoxification treatment was required.

[0124] Table 6. SDS-PAGE results of purified PRN protein

[0125] Sample number Sample Name purity(%) molecular weight kDa 1 Standard products 95 71 2 Purified PRN antigen stock solution 98.2％ 71

[0126] Example 5: Preparation and purification of PT protein

[0127] 1. PT Experimental Materials

[0128] Buffer A-PT: 10 mM PB, pH 6.0;

[0129] Buffer B-PT: 100mM PB, pH 7.0;

[0130] Buffer C-PT: 100mM PB + 0.5M NaCl, pH 7.0;

[0131] Buffer solution D-PT: 20mM PB + 2M urea, pH 6.0;

[0132] Buffer solution E-PT: 20mM PB + 2M urea + 1M NaCl, pH 6.0;

[0133] Buffer F-PT: 20mM PB + 2M urea + 0.5% (g / ml) Triton X-100, pH 6.0;

[0134] Buffer G-PT: 20mM Tris-HCl + 2M urea + 1M NaCl, pH 8.5;

[0135] Buffer H-PT: 20mM Tris-HCl + 2M NaCl, pH 10.0;

[0136] Buffer I-PT: 0.5 mol / L NaCl + 0.05 mol / L PB, pH 8.0.

[0137] 2. Preparation of PT protein toxoids

[0138] 2.1 Preparation and purification of PT protein

[0139] (1) The engineered bacteria OEPRN-PT with the FHA gene knocked out in patent application CN116265584A was used to ferment the bacterial solution. The solution was centrifuged at 4℃ for 30 min, and the supernatant was retained. The supernatant was concentrated with a 10 kDa membrane and impurities were removed by filtering through a filter membrane. The solution was then ultrafiltered and exchanged into buffer A-PT to obtain the ultrafiltrate.

[0140] (2) The CHT column was regenerated by washing with 0.5M NaOH aqueous solution and buffer C-PT sequentially, and then equilibrated to baseline with buffer A-PT at a flow rate of 10 ml / min. The ultrafiltrate was loaded onto the CHT column, and after flow-through, it was washed with buffer A-PT to baseline, then washed with buffer B-PT, and then eluted with buffer C-PT to remove proteins. The PT protein eluent was collected and named CHT-elution buffer.

[0141] (3) The CHT-elution buffer was loaded onto a Capto SP ImpRes chromatography column equilibrated with buffer D-PT. After 5 CV of elution with buffer F-PT to remove endotoxin, the column was washed with 8% buffer E-PT (i.e., 8% (v / v) of buffer E-PT and the remaining 92% (v / v) of buffer D-PT). Then, PT protein was eluted with 20% buffer E-PT (i.e., 20% (v / v) of buffer E-PT and 80% (v / v) of buffer D-PT). This sample was named CEX-elution buffer. The column was then regenerated with buffer H-PT. Subsequently, the CEX-elution buffer was subjected to Capto MMC column chromatography. After elution with buffer E-PT, PT protein was eluted with buffer G-PT. This sample was named MMC-elution buffer, which is the purified PT protein stock solution. The stock solution was analyzed by SDS-PAGE and HPLC.

[0142] SDS-PAGE results show ( Figure 10The PT protein stock solution prepared by engineered bacteria OEPRN-PT had a purity of over 95% compared to the PT standard, indicating the absence of FHA protein and its interference. HPLC results also showed that... Figure 11 The purified PT protein stock solution showed no presence or interference from FHA protein. CHO agglutination assay results ( Figure 12 This also indicates that the PT protein purified by this method has high activity.

[0143] 2.2 Preparation (Detoxification) of PT Protein Toxins

[0144] The MMC-elution buffer was replaced with buffer I-PT to dilute the protein concentration for chemical detoxification. The detoxification steps were as follows: 2 mol / L lysine was added, and a glutaraldehyde aqueous solution was used for the detoxification reaction at 25±1℃ to obtain a glutaraldehyde-detoxified solution. Then, 57.4 mmol / L N-acetyltryptophan aqueous solution and 2.2 mol / L glycine aqueous solution were added to the above glutaraldehyde-detoxified solution, and the reaction was carried out at 25±1℃ for 2.0±0.2 h. After that, a 37 g / L formaldehyde aqueous solution was added, and the reaction was carried out at 37±1℃. After 10 days of reaction, ultrafiltration was performed using a 10 kD ultrafiltration membrane. After concentrating the solution 5-10 times, the detoxified PT protein toxoid stock solution was obtained after filtration.

[0145] Figure 13 The results of the CHO agglutination experiment on the detoxified PT protein toxin stock solution show that even after treatment with a high concentration (100 μg / ml) of PT protein toxin stock solution, the CHO cells remained in good condition and no clustering was observed, indicating that the detoxification process can completely and stably inactivate the purified PT toxin.

[0146] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through some modifications or variations made to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. A pertussis antigen recombinant expression vector comprising an resistance selection gene, an upstream recombinant nucleic acid fragment of a pertussis exotoxin gene, and a downstream recombinant nucleic acid fragment, wherein the upstream and downstream recombinant nucleic acid fragments of the pertussis exotoxin gene are capable of homologous recombination with the upstream and downstream of the pertussis exotoxin gene, respectively.

2. The recombinant expression vector according to claim 1, wherein, The starting vector for the expression vector is pET32a, pET28a, pUC57 or TA, preferably TA.

3. The recombinant expression vector according to claim 1 or 2, wherein, The nucleotide sequence of the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene contains or is a nucleic acid fragment composed of consecutive bases from position m to 986 in the nucleotide sequence shown in SEQ ID NO.2, where m is a natural number ≤ 205; preferably, the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene is a nucleic acid fragment composed of consecutive bases from position 205 to 986 in the nucleoside sequence shown in SEQ ID NO.2; Preferably, the nucleotide sequence of the recombinant nucleic acid fragment downstream of the pertussis exotoxin gene comprises or is a nucleic acid fragment composed of consecutive bases from position 1 to position n in the nucleotide sequence shown in SEQ ID NO.3, where n is a natural number ≥ 870-1090; preferably, the recombinant nucleic acid fragment downstream of the pertussis exotoxin gene is a nucleic acid fragment composed of consecutive bases from position 1 to position 870 in the nucleoside sequence shown in SEQ ID NO.3; Preferably, the nucleotide sequence of the forward primer used to amplify the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene includes SEQ ID NO.8 or is as shown in SEQ ID NO.8; Preferably, the nucleotide sequence of the reverse primer used to amplify the recombinant nucleic acid fragment upstream of the pertussis exotoxin gene includes or is shown in SEQ ID NO. 9; Preferably, the nucleotide sequence of the forward primer used to amplify the resistance selection gene comprises SEQ ID NO.10 or as shown in SEQ ID NO.10; Preferably, the nucleotide sequence of the reverse primer used to amplify the resistance selection gene comprises SEQ ID NO.11 or is as shown in SEQ ID NO.11; Preferably, the nucleotide sequence of the forward primer used to amplify the downstream recombinant nucleic acid fragment of the pertussis exotoxin gene includes SEQ ID NO.12 or is as shown in SEQ ID NO.12; Preferably, the nucleotide sequence of the reverse primer used to amplify the recombinant nucleic acid fragment downstream of the pertussis exotoxin gene includes SEQ ID NO.13 or is as shown in SEQ ID NO.

13.

4. The recombinant expression vector according to any one of claims 1 to 3, wherein, The resistance selection gene is selected from one or more of the following: kanamycin resistance selection gene, tetracycline resistance selection gene, ampicillin resistance selection gene, and chloramphenicol resistance selection gene, preferably a kanamycin resistance selection gene; Preferably, the nucleotide sequence of the kanamycin resistance selection gene is shown in SEQ ID NO.4; the nucleotide sequence of the tetracycline resistance selection gene is shown in SEQ ID NO.5; the nucleotide sequence of the ampicillin resistance selection gene is shown in SEQ ID NO.6; and the nucleotide sequence of the chloramphenicol resistance selection gene is shown in SEQ ID NO.

7.

5. A recombinant pertussis antigen-expressing engineered bacterium, comprising the recombinant expression vector according to any one of claims 1 to 4.

6. The method for preparing the recombinant pertussis antigen-expressing engineered bacteria according to claim 5, comprising transforming wild-type Bordetella pertussis competent cells with the recombinant expression vector according to any one of claims 1 to 4; Preferably, the wild-type Bordetella pertussis is wild-type Bordetella pertussis strain ATCC-BAA-589; Preferably, the conversion is performed by electroconversion; more preferably, the electroconversion includes linearizing the recombinant expression vector and then electroconverting wild-type Bordetella pertussis competent cells at 2200-2800V and 2-8ms.

7. The preparation method according to claim 6, further comprising screening transformed wild-type Bordetella pertussis competent cells using the resistance selection gene.

8. The use of the pertussis antigen recombinant expression vector according to any one of claims 1 to 4 or the pertussis antigen recombinant expression engineered bacteria according to claim 5 in the preparation of pertussis antigen; Preferably, the pertussis antigen is filamentous hemagglutinin and / or pertussis adhesivein.

9. A method for preparing pertussis antigen, comprising fermentation using the pertussis antigen recombinant expression engineered bacteria according to claim 5.

10. The preparation method according to claim 9, wherein, The fermentation includes the following steps: (1) The recombinant pertussis antigen expression engineered bacteria according to claim 5 were inoculated into a Petri dish containing Pougon's blood agar medium and cultured at 35-40°C for 40-72 hours; (2) Inoculate the bacterial cells obtained in step (1) into MSS medium and incubate at 35-40℃ for 12-36 hours; (3) Inoculate the bacterial culture obtained in step (2) into a fermenter containing MSS medium and incubate at 35-40°C for 68-70 hours.

11. A pertussis antigen, prepared by the method according to claim 9 or 10; Preferably, the pertussis antigen is filamentous hemagglutinin and / or pertussis adhesivein.

Images