Attenuated bordetella pertussis recombinant strain as well as construction method and application thereof

Abstract

The invention discloses an attenuated Bordetella pertussis recombinant strain as well as a construction method and application thereof, and belongs to the technical field of microbial genetic engineering. The invention provides an attenuated Bordetella pertussis recombinant strain, compared with a wild type, the toxicity of the gene detoxification Bordetella pertussis toxin expressed by the recombinant strain is reduced by 106 times, and the gene detoxification Bordetella pertussis toxin modified from the CS strain of Bordetella pertussis is proved to have good protectiveness for the first time in a mouse challenge experiment. The compound can be used for preparing products for treating or / and detecting or / and preventing pertussis diseases, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial genetic engineering, and in particular relates to an attenuated Bordetella pertussis recombinant strain and a construction method and application thereof. Background Art

[0002] Pertussis toxin (PT) is a major virulence factor produced by Bordetella pertussis. It is a constitutive A / B bacterial toxin composed of S1, S2, S3, S4, and S5 subunits in a 1:1:1:2:1 ratio. The A monomer, composed of the S1 subunit, possesses ADP-ribosyltransferase activity, blocking the binding of adenylate cyclase to its receptors, leading to an increase in intracellular cAMP (cyclic adenosine monophosphate) and numerous biological effects, such as histamine sensitization, leukocytosis, and insulin secretion. The B oligomer, composed of S2, S3, S4, and S5, is a non-toxic polymer that binds to various receptors on the surface of eukaryotic cells, transporting the S1 subunit and exerting its physiological toxicity. The B oligomer also activates and regulates immune responses.

[0003] Pertussis toxin is a key component of currently available vaccines, and antibodies against it are associated with protection against pertussis in children. However, due to the biotoxicity of its S1 subunit, pertussis toxin must be detoxified before use in vaccine preparation. Currently, methods for detoxifying pertussis toxin (PT) are primarily chemical and genetic. Chemical detoxification employs reagents such as formaldehyde, glutaraldehyde, and hydrogen peroxide. Chemical detoxification modifies and binds to reactive amino acid groups on the protein's surface, thereby removing biological activity. Due to the random nature of chemical reactions and the disruption of the protein's multidimensional structure, which in turn affects its immunogenicity, controlling the chemical detoxification process has long been challenging in product development and has also impacted vaccine efficacy. Genetic detoxification, on the other hand, targets the ADP-ribosyltransferase activity of the S1 subunit by mutating the two amino acids R9K / E129G. These two mutations significantly reduce the S1 subunit's enzymatic activity—and, consequently, its biotoxicity—while essentially preserving the structural properties of native PT, maximizing the preservation of surface antigenic determinants and resulting in improved protection. Moreover, the stability of gene detoxification also solves the controllability problem of chemical detoxification process.

[0004] Currently, the genetic detoxification of PT is basically derived from the European and American pertussis Tohama strain (BAA-589), including the genetically attenuated diphtheria, pertussis and tetanus vaccine that has been used in adolescents and adults. However, the CS strain used in the production of pertussis vaccine in China has a different amino acid at position 194 in the S1 subunit compared to the Tohama strain (methionine at position 194 in the CS strain and isoleucine at position 194 in the Tohama strain), and there are also significant differences between the domestic clinical epidemic strains and the foreign epidemic strains. So whether PT completes mutations at the same site (such as R9K and / or E129G) can also have a similar protective effect to the genetically detoxified PT based on the Tohama strain is worthy of real-world research. According to publicly reported data, the only company currently carrying out genetic attenuation modification for the CS strain of Bordetella pertussis is Wuhan Institute of Biological Products Co., Ltd., which used a sequence fragment with a resistance gene in its patent published in 2023 (CN 117844719 A) to construct a recombinant strain of PT genetically detoxified by non-scarless gene editing technology. Although it has been successfully proven to reduce the toxicity of PT, there is no direct evidence to prove that it has effective protection; moreover, a new functional group sequence - a kanamycin resistance sequence - was additionally introduced into the constructed strain, which not only introduced resistance genes into subsequent possible production strains, but also brought additional risks to production; and the expression level of genetically detoxified PT (gPT) was also very low, less than 0.8 mg / L fermentation liquid, making it difficult to achieve large-scale production.

[0005] Therefore, constructing a new gene-free, highly expressed, gene-free strain based on the CS strain of Bordetella pertussis used in the production of pertussis vaccine in China will have significant scientific significance and social value, and will lay the foundation for the research and development of new pertussis vaccines. Summary of the Invention

[0006] In order to solve the above problems existing in the prior art, the purpose of the present invention is to provide an attenuated recombinant strain of Bordetella pertussis and a construction method and application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a method for constructing a recombinant strain of Bordetella pertussis, the method comprising the following steps: 1) Construction of the knockout plasmid pJZ-G-UD containing the upstream and downstream homologous recombination arms of the pertussis toxin S1 subunit gene and the knock-in plasmid pJZ-G-mS1 containing the upstream and downstream homologous recombination arms of the pertussis toxin S1 subunit gene and the pertussis toxin S1 subunit double-site mutation gene; The structure of the knockout plasmid pJZ-G-UD is: plasmid replication origin p15A ori- plasmid transfer origin oriT- sacB Promoter- sacB -Resistance genes- galkPromoter- galk -lac promoter-upstream (5') homology arm-downstream (3') homology arm, wherein the direction of the resistance gene and the plasmid replication origin p15A ori is opposite to that of other genes on the plasmid; The knock-in plasmid pJZ-G-mS1 has the structure of plasmid replication origin p15A ori- plasmid transfer origin oriT- sacB Promoter- sacB -Resistance genes- galk Promoter- Galk -lac promoter-upstream (5') homology arm-pertussis toxin S1 subunit double-site mutation gene-downstream (3') homology arm, wherein the direction of the resistance gene and the plasmid replication origin p15A ori is opposite to that of other genes on the plasmid; 2) Preparation of wild-type Bordetella pertussis competent cells; 3) Transforming the knockout plasmid pJZ-G-UD constructed in step 1) into the wild-type Bordetella pertussis competent cells obtained in step 2), resuscitating the cells, and subjecting them to forward screening and reverse screening to obtain a recombinant Bordetella pertussis strain ΔS1 lacking the pertussis toxin S1 subunit gene; 4) Preparation of competent cells of the recombinant Bordetella pertussis strain ΔS1; 5) The knock-in plasmid pJZ-G-mS1 constructed in step 1) was transformed into competent cells of the recombinant Bordetella pertussis ΔS1 strain obtained in step 4), and the cells were revived and subjected to forward screening and reverse screening to obtain a recombinant Bordetella pertussis strain with a double-site mutation in the S1 subunit.

[0008] Furthermore, the Bordetella pertussis is the Bordetella pertussis CS strain, and the resistance gene is the gentamicin resistance gene GmR.

[0009] Furthermore, the nucleotide sequence of the knockout plasmid pJZ-G-UD in step 1) is SEQ ID No. 3, and the nucleotide sequence of the knockin plasmid pJZ-G-mS1 is SEQ ID No. 4.

[0010] Furthermore, the method for preparing the competent cells in step 2) is to inoculate the revived wild-type Bordetella pertussis CS strain into Stainer-Scholte liquid culture medium and culture until OD 550nm =2.8~3.2, collect the bacteria by centrifugation, wash the bacteria with sterile water at 2~8℃, wash twice with 10% glycerol at 2~8℃, and then resuspend the bacteria with 10% glycerol at 2~8℃.

[0011] Furthermore, in step 3), the conversion is performed by electroporation, and the parameters of the electroporation are set as the distance between the electrodes of the electric shock cup is 0.1-5 mm, the capacitance is 15-35 μF, the resistance is 100-300 Ω, the voltage is 2000-3000 V, and the electroporation time does not exceed 5.5 ms.

[0012] Furthermore, in step 3), the parameters of the electroporation are set as follows: the distance between the electrodes of the shock cup is 1 mm, the capacitance is 25 μF, the resistance is 200 Ω, the voltage is 2500 V, and the electroporation time is no more than 5 ms.

[0013] Furthermore, in step 3), the forward screening is performed by spreading the revived bacterial solution on a Bordet-Gengo solid culture medium plate containing gentamicin and culturing at 36±1°C for 5 to 7 days; and the reverse screening is performed by spreading the bacterial solution obtained by the forward screening on a Bordet-Gengo solid culture medium plate containing sucrose and 2-deoxygalactose and culturing at 36±1°C for 5 to 7 days.

[0014] Furthermore, in step 3), the carbon-coated Bordet-Gengo solid culture medium plate containing gentamicin for forward screening is a carbon-coated Bordet-Gengo solid culture medium plate containing 50 ppm gentamicin; and the carbon-coated Bordet-Gengo solid culture medium plate containing sucrose and 2-deoxygalactose for reverse screening is a carbon-coated Bordet-Gengo solid culture medium plate containing 15% sucrose and 0.1% 2-deoxygalactose.

[0015] Furthermore, the method for preparing the competent cells in step 4) is to inoculate the revived Bordetella pertussis recombinant strain ΔS1 into Stainer-Scholte liquid culture medium and culture until OD 550nm =2.8~3.2, collect the bacteria by centrifugation, wash the bacteria with sterile water at 2~8℃, wash twice with 10% glycerol at 2~8℃, and then resuspend the bacteria with 10% glycerol at 2~8℃.

[0016] Furthermore, in step 5), the conversion is performed by electroporation, and the parameters of the electroporation are set as the shock cup electrode spacing of 0.1-5 mm, the capacitance of 15-35 μF, the resistance of 100-300 Ω, the voltage of 2000-3000 V, and the electroporation time of no more than 5.5 ms.

[0017] Furthermore, in step 5), the parameters of the electroporation are set as follows: the distance between the electrodes of the shock cup is 1 mm, the capacitance is 25 μF, the resistance is 200 Ω, the voltage is 2500 V, and the electroporation time is no more than 5 ms.

[0018] Furthermore, in step 5), the forward screening is performed by spreading the revived bacterial solution on a Bordet-Gengo solid culture medium plate containing gentamicin and culturing at 36±1°C for 5 to 7 days; and the reverse screening is performed by spreading the bacterial solution obtained by the forward screening on a Bordet-Gengo solid culture medium plate containing sucrose and 2-deoxygalactose and culturing at 36±1°C for 5 to 7 days.

[0019] Furthermore, in step 5), the carbon-coated Bordet-Gengo solid culture medium containing gentamicin for forward screening is a carbon-coated Bordet-Gengo solid culture medium plate containing 50 ppm gentamicin; and the carbon-coated Bordet-Gengo solid culture medium plate containing sucrose and 2-deoxygalactose for reverse screening is a carbon-coated Bordet-Gengo solid culture medium plate containing 15% sucrose and 0.1% 2-deoxygalactose.

[0020] The carbon-coated Bordet-Gengo solid medium plates containing 15% sucrose and 0.1% 2-deoxygalactose (DOG) used in the reverse screening were obtained after extensive screening. The combined effects of 15% sucrose and 0.1% DOG significantly increased the secondary homologous recombination rate, from 20% with 0.1% DOG alone and less than 10% with 15% sucrose alone to over 95%. This effectively reduced the single DOG concentration while maintaining a high recombination rate.

[0021] The present invention also provides a recombinant strain of Bordetella pertussis obtained according to the above construction method.

[0022] The present invention also provides the use of the above-mentioned Bordetella pertussis recombinant strain in preparing products for treating and / or preventing pertussis disease.

[0023] Furthermore, the product is a pertussis vaccine or a diphtheria, tetanus and pertussis vaccine.

[0024] The present invention has the following beneficial effects: 1) The recombinant strain of Bordetella pertussis prepared in the present invention is constructed by electroporation to introduce a suicide plasmid carrying the target nucleotide sequence to complete the editing and transformation of the target gene. Compared with the traditional method of introducing the recombinant plasmid by binding to Escherichia coli, the nucleic acid introduction method of the present invention avoids the introduction and contamination of Escherichia coli. At the same time, the introduced plasmid is a suicide plasmid, which will not accumulate and replicate in the target bacteria, and will gradually disappear with the metabolism and propagation of the strain, and will not pollute the genetic background. 2) The recombinant strain of Bordetella pertussis prepared in the present invention, whether it is an S1 subunit double-site mutation (R9K / E129G) strain or an S1 subunit full-length deletion strain, will not introduce resistance genes, other functional genes and additional nucleotide sequences into its genome, and will not increase the risk to strain stability, biosafety and subsequent production; 3) The gene-detoxified PT expressed by the recombinant strain with a double-site mutation of the S1 subunit prepared in the present invention is 10% less toxic than the WHO second-generation pertussis toxin standard 15 / 126. 6 The results of this study demonstrate that the gPT derived from the genetically modified Bordetella pertussis CS strain exhibits excellent protective efficacy, meeting the requirements of the Chinese Pharmacopoeia. Furthermore, the recombinant strain with double mutations in the S1 subunit prepared in this invention exhibits gPT expression levels consistent with those reported in the literature for a recombinant strain derived from the Tohama pertussis strain (BAA-589) produced by Bionet, thus meeting the requirements for large-scale production. This research lays the foundation for research into the mechanism of action of the PT protein of Bordetella pertussis CS strain and the development of genetically detoxified pertussis vaccines.

[0025] In summary, the present invention provides a recombinant strain of attenuated Bordetella pertussis. The gene-detoxified pertussis toxin expressed by the recombinant strain of the present invention is 10% less toxic than the wild type. 6 times, and in the mouse challenge experiment, it was proved for the first time that the genetically detoxified pertussis toxin derived from the modified CS strain of Bordetella pertussis has good protective properties, meets the relevant requirements of the "Chinese Pharmacopoeia", and can be used to prepare products for the treatment and / or detection and / or prevention of pertussis diseases, and has good application prospects.

[0026] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for the terms in this document apply to the terms throughout the specification; for terms not specifically defined herein, they should be given the meaning that a person skilled in the art would give them based on the disclosure and context.

[0027] The term "scarless gene editing" refers to the modification of a target gene without the introduction of additional DNA sequences. This modification is typically performed through homologous recombination: using upstream and downstream homologous arms at specific sites on either side of the target gene to be edited, forward screening is used to introduce the target gene nucleotide sequence and the screening gene nucleotide sequence into the genome. Reverse screening is then used to obtain a secondary recombinant strain that has deleted the screening gene nucleotide sequence but retained the target gene nucleotide sequence. Scarless editing allows for the stacking of multiple genetic modifications. In contrast, when scarring gene editing is performed using non-scarless methods, for example, resistance replacement methods, resistance genes are introduced simultaneously with successful modification.

[0028] The term "homologous arms" refers to flanking sequences upstream and / or downstream of the specific site where the target gene is to be inserted or replaced, or sequences that have at least 95% sequence identity with the flanking sequences. The length of the homology arms can generally be several hundred bp, or even more than 1000 bp.

[0029] The term "positive screening" refers to identifying integration of exogenous sequences into the B. pertussis genome by positively screening for the presence and / or expression of genes.

[0030] The term "counter-screening" refers to identifying deletion of a screening nucleotide sequence from the B. pertussis genome by the absence and / or non-expression of a counter-screening gene.

[0031] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0032] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the structure of pJZ-G-UD plasmid and pJZ-G-mS1 plasmid.

[0034] Figure 2 This is the PCR verification result of the recombinant strain grown by pJZ-G-UD transformation. sacB Primer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0035] Figure 3 The PCR verification results of the secondary recombinant strain grown by pJZ-G-UD transformation are shown in Figure 2. sacBPrimer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0036] Figure 4 This is the PCR verification result of the recombinant strain grown by pJZ-G-mS1 transformation. sacB Primer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0037] Figure 5 The PCR verification results of the secondary recombinant strain grown by pJZ-G-mS1 transformation are shown in Figure 2. sacB Primer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0038] Figure 6 Figure 2 shows the growth curves of the recombinant strain mS1 and the wild-type Bordetella pertussis CS strain (WT).

[0039] Figure 7 The PT protein time-effect curves of the recombinant strain mS1 and the wild-type Bordetella pertussis CS strain (WT).

[0040] Figure 8 Comparative capillary electrophoresis spectra of wild-type pertussis toxin (red) and genetically detoxified pertussis toxin (blue) proteins.

[0041] Figure 9 This is a graph showing the different clustering degrees of CHO cells. DETAILED DESCRIPTION

[0042] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0043] The following experiments, where no temperature is specified, are reactions conducted at room temperature, which is 25±5°C.

[0044] The Bordetella pertussis CS strain used in the following examples has a deposit number of CMCC 58003. CHO cells (ATCC-CCL-61) were provided by the Product Processing Laboratory of Chengdu Institute of Biological Products Co., Ltd. Both Stainer-Scholte Broth (SSM) and Bordet-Gengou (BG) medium were prepared and provided by the Culture Medium Laboratory of Chengdu Institute of Biological Products Co., Ltd. CHO cell culture medium DMEM / F12 (1:1) (Cat. No. 11330032) was purchased from Gibco. Mice used in efficacy experiments were provided by the Animal Laboratory of Chengdu Institute of Biological Products Co., Ltd.

[0045] R9K indicates that the arginine (R) at position 9 on the pertussis toxin S1 subunit mutates to lysine (K), and E129G indicates that the glutamic acid (E) at position 129 on the pertussis toxin S1 subunit mutates to glycine (G).

[0046] Example 1: Preparation of attenuated Bordetella pertussis recombinant strain 1. Preparation of a recombinant strain of Bordetella pertussis lacking the PT-S1 subunit 1.1 Construction of plasmids pJZ-G-UD and pJZ-G-mS1 Through gene synthesis and enzyme ligation, sacB Suicide plasmid insertion of genes galk Sequence (with promoter), so that the plasmid has gentamicin resistance, sacB and galk gene function, named pJZ-G; galk The gene nucleotide sequence information is shown in SEQ ID No.5: Using the restriction enzyme sites SacI and BamHI in the plasmid multiple cloning region as the target fragment ligation sites, the 5' homology arm (950 bp)-3' homology arm (1650 bp) of the PT-S1 subunit gene was integrated into the plasmid pJZ-G by gene synthesis to obtain the plasmid pJZ-G-UD; the nucleotide sequence information of the 5' homology arm (950 bp)-3' homology arm (1650 bp) is shown in SEQ ID No. 6: The plasmid pJZ-G-UD contains ori-p15A, gentamicin resistance gene ( GmR )、 sacB and galk The anti-screening gene, the multiple cloning site region contains 5' homology arms and 3' homology arms, and its structure is as follows Figure 1 shown.

[0047] Plasmid pJZ-G-UD was used to knock out the target gene to be edited, the S1 subunit.

[0048] The target gene (mS1) nucleotide sequence was inserted between the upstream (5') homology arm (950bp) and the downstream (3') homology arm (1650bp) in the multicloning region of the plasmid pJZ-G-UD by gene synthesis and enzyme ligation to obtain the plasmid pJZ-G-mS1. Theoretically, the mS1 gene can be any target gene that can be introduced. In this embodiment, the mS1 nucleotide sequence specifically refers to the sequence after mutation of two specific sites of the target gene to be edited - the pertussis toxin S1 subunit. The nucleotide sequence information of 5' homology arm (950bp)-mS1-3' homology arm (1650bp) is shown in SEQ ID No.7. The plasmid pJZ-G-mS1 contains ori-p15A, gentamicin resistance gene ( GmR )、 sacB and galk The anti-screening gene, the multiple cloning site region contains 5' homology arm-mS1-3' homology arm, and its structure is as follows Figure 1 shown.

[0049] Using plasmids pJZ-G-UD and pJZ-G-mS1, double-site mutation of the S1 subunit was achieved by knocking out the target gene to be edited and knocking in the target gene, respectively.

[0050] 1.2 Preparation of competent cells Wild-type Bordetella pertussis CS strain stored at -80°C was taken out of the refrigerator, rapidly thawed in a biosafety cabinet, inoculated into 5 mL of SSM medium, and cultured at 36 ± 1°C and 250 × rpm for 24 hours. The recovered bacterial solution was inoculated into 50 mL of SSM medium, and the final concentration of the bacteria after inoculation was adjusted to about 200 million CFU / mL. The culture was carried out at 36±1℃ and 250×rpm for 22-24 hours. The OD 550 = around 3.0; In a biosafety cabinet, collect the bacterial solution in a 50 mL sterile centrifuge tube and incubate at 4°C, 4000 × g Centrifuge for 15 minutes under the same conditions to collect the bacteria; Resuspend the cells with 25 mL of sterile double-distilled water cooled in an ice water bath and wash the cells by gently blowing and stirring repeatedly with a pipette and a sterile pipette. g Centrifuge for 15 minutes under the same conditions and collect the bacteria again; Resuspend the bacteria in 10 mL of 10% (V / V) glycerol pre-cooled in an ice water bath, quickly and gently blow off the bacteria with a pipette and a sterile pipette, and place them in ice water for 10 minutes, then incubate at 4°C, 4000× g Centrifuge for 15 minutes under the same conditions to collect the bacteria, and perform this step twice; Quickly resuspend and mix the cells with 1 mL of 10% (V / V) glycerol pre-cooled in an ice water bath, then aliquot 100 μL per tube and store at -80°C for later use or directly transfer to an ice water bath pre-cooled electroporation cuvette for electroporation.

[0051] 1.3 Electroporation and plate screening 1 Take out 100 μL of competent cells prepared in step 1.2 from the -80℃ freezer and thaw on ice. At the same time, place the electroporation cuvette (1mm gap) and the plasmid pJZ-G-UD prepared in step 1.1 on ice and pre-cool for 5 minutes. In a biosafety cabinet, add 1 μg of plasmid pJZ-G-UD to 100 μL of competent cells, mix well, and transfer to an electroporation cuvette. Operate in an ice-water bath throughout the process. Set the parameters to 2500V, 25μF, and 200Ω. Ensure that the bacterial and plasmid mixture is at the bottom of the electroporation cup. Wipe off the condensed water outside the electroporation tank, insert the electroporation cup, and start the electric pulse. After the electroporation, remove the sample pool as soon as possible, immediately add 1 mL of SSM medium preheated at 37°C, transfer to a sterile centrifuge tube, and resuscitate on a constant temperature shaker at 36±1°C and 250× rpm for 12 h. The bacterial solution was spread on a carbon-coated BG resistance plate containing 50 ppm gentamicin, cultured at 36±1°C for 5-7 days, and strains with positive gentamicin resistance were screened.

[0052] 1.4 Detection of gene level of recombinant strains 1 Use a sterile pipette tip to pick up the single colony grown on the gentamicin resistance screening plate and streak it on a new gentamicin resistance BG plate, and dip the remaining bacteria on the pipette tip into 20uL sterile water as a PCR verification template; use plasmids sacB Primers and S1 internal primers were used to verify whether homologous recombination had occurred between the plasmid and the bacterial genome. Furthermore, primers specific for integration of the up-end homology arm and the down-end homology arm were used to verify whether the plasmid integrated via single crossover of the upstream homology arm or the downstream homology arm. The primers used for PCR verification are listed in Table 1.

[0053] Table 1 Knockout recombination verification primer list The results of PCR verification of the primary recombinant strain grown by pJZ-G-UD transformation are as follows Figure 2 As shown, Figure 2 A uses primers Text Sacb s, Text Sacb as to randomly select 1 # -8 # A 951 bp fragment was cloned from each strain ( sacB sequence), and Figure 2 B uses primers Text S1wb s, Text S1wb as in 1 # -8 # Two fragments of 319 bp and 1129 bp were cloned simultaneously in the strain, indicating that 1 # -8 # All cloned bacteria underwent single crossover of homology arms, and the sequence on the plasmid was integrated into the genome; Figure 2 C used the up end homology arm integration specific primers Text up s and Text up as to clone a 1150 bp fragment. Figure 2 D used the down end homology arm integration specific primer to clone a 2660 bp fragment. The above results proved that 1 # -8 # The strains were recombined by single crossover through the upstream homology arm. The first homologous recombination of Bordetella pertussis recombinant strain was named UP-sacB + -galk + 1 # -8 # The template for lane 9 is water, the template for lane 10 is the pJZ-G-UD plasmid, the template for lane 11 is the pJZ-G-mS1 plasmid, and the template for lane 12 is the wild-type pertussis strain.

[0054] 1.5 Plate Screening 2 Randomly select a plant UP-sacB + -galk + After the bacteria were amplified in SSM medium, they were re-diluted or streaked onto carbon-finished BG medium with sucrose (15%) and DOG (0.1%) concentrations according to different dilution multiples and cultured at 36±1℃ for 5-7 days. If the second homologous recombination does not occur in the upstream or downstream homologous recombination arms of the S1 subunit, sacB + -galk + Under the action of the gene, the bacteria will metabolize sucrose and DOG, resulting in a lethal effect and will not be able to grow on the BG plate; if the homologous recombination arm undergoes a second homologous recombination, the genome will be lost. sacB + -galk + After the gene is inserted, the bacteria will be able to grow on BG plates.

[0055] 1.6 Detection of gene level of recombinant strain 2 According to the operation procedure in 1.5, PCR verification was performed on the single clone grown on the reverse screening plate. The results were as follows: Figure 3 Middle 1 # -18# The strain results are shown in Figure 1. Lanes 1-14 show single colonies grown on sucrose / DOG carbon powder solid plates. Lane 15 is water, lane 16 is pJZ-G-UD, lane 17 is pJZ-G-mS1, and lane 18 is a wild-type pertussis strain. Figure 3 A in 1 # -14 # The primers Text Sacb s and Text Sacb as did not amplify any bands in the strain, indicating that a second homologous recombination occurred under the action of sucrose and the DOG anti-screening gene, successfully losing the sequences in the non-upstream and downstream homologous recombination arm regions on the plasmid. Figure 3 In B, primers Text S1wb s and Text S1wb as were used for lane 1. # , 3 # , 4 # , 6 # , 7 # , 8 # , 10 # and 13 # Only 319 bp were amplified, combined with Figure 3 The PCR results of the up-end and down-end specific primers of the corresponding monoclonal strains of C / D indicate that the second homologous recombination occurred in the downstream homologous recombination arm, and the recombinant strain of Bordetella pertussis with PT S1 subunit deletion was successfully obtained and named ΔS1; while for lane 2 # , 5 # and 9 # Only 1129 bp was amplified, indicating that the second homologous recombination occurred in the upstream homologous recombination arm and the strain returned to the wild type state. # , 12 # and 14 # The fact that 319bp and 1129bp can be amplified simultaneously indicates that the selected monoclonal strain is impure and that there are strains in which a second homologous recombination occurs both upstream and downstream.

[0056] 1.7 Preservation of recombinant strains The verified monoclonal strain ΔS1 was spread on a carbon powder BG solid plate and grown at 36±1°C for 5-7 days. All bacteria were scraped off the plate and transferred to SSM medium. They were cultured at 36±1°C and 250×rpm for 24-28 hours to an OD value of approximately 4-5. A 40% glycerol solution was prepared and sterilized under high pressure. The bacterial solution was mixed with the 40% glycerol solution in a 1:1 ratio, and after mixing evenly, the mixture was divided into sterile cell cryopreservation tubes and stored in a -80°C refrigerator.

[0057] 2. Preparation of a recombinant strain of Bordetella pertussis with a double mutation in the PT-S1 subunit (R9K / E129G) 2.1 Construction of knock-in plasmid pJZ-G-mS1 Using the restriction enzyme sites SacI and BamHI in the multiple cloning site region of plasmid pJZ-G as the target fragment ligation sites, the 5' homology arm (950 bp) of the PT-S1 subunit gene, the S1 subunit double-site mutant gene, and the 3' homology arm (1650 bp) of the PT-S1 subunit gene were integrated into the pJZ-G plasmid by gene synthesis to obtain plasmid pJZ-G-mS1; the nucleotide sequence information of 5' homology arm (950 bp)-mS1-3' homology arm (1650 bp) is shown in SEQ ID No. 7: The plasmid pJZ-G-mS1 contains ori-p15A, gentamicin resistance gene (GmR), sacB and galk The anti-screening gene, the multiple cloning site region contains a 5' homology arm, a double-site mutation (R9K / E129G) sequence of the S1 subunit and a 3' homology arm, and its structure is as follows: Figure 1 shown.

[0058] 2.2 Preparation of competent cells Take the PT-S1 subunit-deficient Bordetella pertussis ΔS1 strain out of the -80°C freezer and prepare competent cells as in step 1.2.

[0059] 2.3 Electroporation and plate screening 1 The competent cells prepared in step 2.2 and the plasmid prepared in step 2.1 were electroporated according to the procedure in step 1.3 and the bacterial solution was spread on a carbon-finished BG resistance plate containing 50 ppm gentamicin. The plates were cultured at 36±1°C for 5-7 days to screen out strains that were positive for gentamicin resistance.

[0060] 2.4 Detection of gene level of recombinant strains 1 According to the operation procedure in 1.4, PCR verification was performed on the single clones grown on the positive screening plate. The primers used for PCR verification are shown in Table 2.

[0061] Table 2 Primers for S1 subunit double-site mutation (mS1) knock-in recombination verification The results of PCR verification of the primary recombinant strain grown by pJZ-G-mS1 are as follows Figure 4 As shown, Figure 4 A uses primers Text Sacb s, Text Sacb as to randomly select 1 # -6 # A 951 bp fragment was cloned from each strain ( sacB sequence), and Figure 4 B uses primers Text S1wb s, Text S1wb as in 1# -6 # Two fragments of 319 bp and 1129 bp were cloned simultaneously in the strain, indicating that 1 # -6 # All cloned bacteria underwent single crossover of homology arms, and the sequence on the plasmid was integrated into the genome; Figure 4 C uses primers Text up s, Text up as in 5 # A 1960 bp fragment was cloned in the strain, indicating that the first homologous recombination occurred in the upstream homologous recombination arm of strain 5#; Figure 4 D uses primers Text down s, Text down as in 1 # , 2 # , 3 # , 4 # and 6 # A 2660 bp fragment was cloned from the strain, indicating that 1 # , 2 # , 3 # , 4 # and 6 # The strain underwent the first homologous recombination in the downstream homologous recombination arm. The first homologous recombination of Bordetella pertussis ΔS1 strain was named UP- or DOWN-sacB + -galk + ΔS1 1 # -6 # The PCR templates for lanes 1-6 are single colonies grown on GM50ppm carbon powder solid plates, the template for lane 7 is water, the template for lane 8 is pJZ-G-UD plasmid, the template for lane 9 is pJZ-G-mS1 plasmid, and the template for lane 10 is the wild-type pertussis strain.

[0062] 2.5 Plate screening 2 Choose any one UP-sacB + -galk + ΔS1 strains and DOWN-sacB + -galk + ΔS1 After the strain was amplified in SSM medium, it was restreaked and gradually spread on carbon-finished BG medium with sucrose (15%) + DOG (0.1%) concentrations and cultured at 36±1℃ for 5-7 days.

[0063] 2.6 Detection of gene level of recombinant strains 2 According to the operation procedure in 2.5, PCR verification was performed on the single clone grown on the reverse screening plate. The results were as follows Figure 5 As shown. UP-sacB + -galk + ΔS1 A total of 6 single clones were picked out ( Figure 5 Lane A, 1-6); DOWN-sacB + -galk + ΔS1 A total of 14 single clones were picked out ( Figure 5 Lane A, 7–20). Figure 5 A in 1 # -20 # The primers Text Sacb s and Text Sacb as did not amplify any bands in the strain, indicating that a second homologous recombination occurred under the action of sucrose and the DOG anti-screening gene, successfully losing the sequences in the non-upstream and downstream homologous recombination arm regions on the plasmid. Figure 5 B in 1 # -6 # The first homologous recombination of the strain occurred in the upstream homologous recombination arm, and the primers Text S1wb s and Text S1wb as were used for lane 1. # , 2 # , 3 # and 5 # 1129 bp were amplified, indicating that the second homologous recombination of the strain occurred in the downstream homologous recombination arm, and the mS1 gene was successfully inserted into the genome; # and 6 # 319bp was amplified, indicating that the second homologous recombination of the strain occurred again in the upstream homologous recombination arm, and the strain was restored to the ΔS1 strain before the resistance positive screening. # -20 # The first homologous recombination of the strain occurred in the downstream homologous recombination arm, and the primers Text S1wb s and Text S1wb as were used for lane 7. # , 9 # 、11 # , 13 # 、17 # and 20 # 1129 bp were amplified, indicating that the second homologous recombination of the strain occurred in the upstream homologous recombination arm, and the mS1 gene was successfully inserted into the genome; # , 10 # , 12 # 、14 # , 15 # , 16 # and 19 # Only 319 bp were amplified, indicating that the second homologous recombination occurred again in the downstream homologous recombination arm, and the strain was restored to the ΔS1 strain before the resistance positive screening. Figure 5 In CD, primers Text up s / Text up as and Text down s / Text down as were used to target 1 # -6 # and 7 # -20 # The molecular weight difference of the cloned fragments of the strains also proved that the recombinant strain 1 # , 2 # , 3 # , 5 # , 7 # , 9 # 、11 # , 13 # 、17 # and 20 # The insertion site of the mS1 sequence is between the upstream and downstream homology arms. A recombinant strain of Bordetella pertussis harboring a double-site mutation (R9K / E129G) in the PT S1 subunit was successfully obtained and named mS1. The PCR templates for lanes 1-20 are single colonies grown on sucrose / DOG carbon powder plates, the template for lane 21 is water, the template for lane 22 is the pJZ-G-UD plasmid, the template for lane 23 is the pJZ-G-mS1 plasmid, and the template for lane 24 is the wild-type pertussis strain. 2.7 Preservation of recombinant strain mS1 The verified monoclonal strain mS1 was spread on a carbon powder BG solid plate and grown at 36±1°C for 5-7 days. All bacteria were scraped off the plate and transferred to SSM medium. They were cultured at 36±1°C and 250×rpm for 24-28 hours to an OD value of approximately 4-5. A 40% glycerol solution was prepared and sterilized under high pressure. The bacterial solution was mixed with the 40% glycerol solution in a 1:1 ratio, and after mixing evenly, the mixture was divided into sterile cell cryopreservation tubes and stored in a -80°C refrigerator.

[0064] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0065] Experimental Example 1: Growth curve, gPT expression, cytotoxicity and protective efficacy of recombinant strain mS1 1.1 Growth curve of recombinant strain mS1 and expression curve of gene-detoxified PT protein (gPT) Randomly select different numbers of Bordetella pertussis recombinant strain mS1 and wild-type CS strain and inoculate them into 50mL SSM medium in 250mL shake flasks. The final concentration of the inoculum is controlled to 200 million CFU / mL. The shake flasks are placed on a constant temperature shaker and cultured continuously at 36±1℃ and 250×rpm. Bacterial samples are taken at 0h, 16h, 19h, 22h, 25h, and 28h to detect the OD value of the bacterial solution. 550 The nm value is plotted into a curve, and the result is as follows Figure 6As shown, the growth rates of the various mS1 recombinant strains were similar to those of the wild-type pertussis CS strain.

[0066] Bacterial samples were collected at 0h, 16h, 19h, 22h, 25h, and 28h, centrifuged at room temperature and 12000×rpm for 8 minutes, and the culture supernatant was collected. The samples were diluted 50 times and 100 times, respectively, and tested using the PT monoclonal antibody double sandwich ELISA method. The PT protein detection range was 1.5625-200ng / mL, and the standard curve of the sample corresponding to the value in this range was multiplied by the dilution multiple to obtain the arithmetic mean, and the gPT content in the collected culture supernatant was obtained. The time-effect curve was drawn as shown in the following figure. Figure 7 As shown in the results, the gPT protein content secreted by the recombinant strain of Bordetella pertussis increased with the culture time during continuous culture, with the maximum value being approximately 2800-3000 ng / mL, which is basically consistent with the gPT expression level of the strain obtained by Bionet based on the genetic modification of the Tohama pertussis strain reported in the literature [1].

[0067] 3.2 Capillary electrophoresis (CE) analysis of the structural integrity of the PT protein expressed by the recombinant strain mS1 The supernatants of the recombinant strain mS1 and the wild-type strain cultured for 28 hours in step 3.1 were purified by column chromatography, gPT and PT, respectively. The protein subunits were quantified by the Lowry method and analyzed by capillary electrophoresis.

[0068] At least 200 μg of gPT and PT samples were collected and concentrated to 2 mg / mL using a 10 kD ultrafiltration centrifuge tube. The buffer was then exchanged twice with 100 mM Tris and 1% SDS. The sample concentration was maintained at 2 mg / mL. After reduction with a defined proportion of β-mercaptoethanol in a fume hood, the sample was transferred to a sample vial and loaded. Capillary electrophoresis conditions were an injection voltage of -10 kV, a migration voltage of -15 kV, a detection wavelength of 220 nm, and a reference wavelength of 360 nm.

[0069] The results are as follows Figure 8 As shown in the figure, both gPT and PT proteins have five independent peaks, which are S1 to S5 in order of peak elution time, which is consistent with the characteristic peak shape of pertussis toxin capillary electrophoresis in the literature [2]. The peak elution time of each subunit of gPT and PT is basically the same.

[0070] 3.3 Cytotoxicity assay of the PT protein of the recombinant strain mS1 The in vitro toxicity of the PT protein of the recombinant strain mS1-8 was evaluated using a CHO cell clustering assay.

[0071] CHO cells that have grown into a dense monolayer were digested with trypsin and then pipetted with culture medium until the cells were suspended in a single layer and diluted to a concentration of 2×10 4 / mL, added to a 96-well plate, 100uL / well, incubated at 37℃ for 12-16h before use; the WHO second-generation pertussis toxin standard 15 / 126 was diluted to one activity unit (1IU / ml) as the standard, the cell culture medium was used as the negative control, the wild-type pertussis toxin PT obtained by column chromatography purification was used as the positive control, and the recombinant strain mS1-8 was cultured in different batches in a fermenter, and the supernatant was purified by column chromatography to obtain different batches of gPT as the test samples; in the dilution plate, 125µL of cell culture medium was used as the diluent, 250µL of each of the above samples was added to the first column of wells, and after stepwise dilution, all were transferred to the pre-incubated adherent cell plate. After incubation at 37℃ for 45-48h, the 96-well plate was placed under a microscope to observe the cell clustering. If no cells clustered, it was judged as no clustering "-", if less than 50% of cells clustered, it was judged as suspicious clustering "±", if 50% or more of cells clustered, it was judged as clustering "+", and if 100% of cells clustered, it was judged as obvious clustering "++". Figure 9 Sample toxicity results were calculated using the concentration calculated using a 15 / 126 final cluster well dilution factor for each 96-well plate. The calculation formula is: Sample activity (IU / mg) = International standard activity (IU / mL) / Sample cluster endpoint concentration (mg / mL). The gPT cytotoxicity test results are shown in Table 3. Activity result 1 represents the first round of testing, and activity result 2 represents the second round of testing. gPT (lot number 20250102) was not included in the first round of testing.

[0072] Table 3 Cytotoxicity test results of different batches of gPT Therefore, compared with the wild-type pertussis toxin PT, the in vitro toxicity of gPT protein expressed by different batches of recombinant strains was reduced to 0.0001%, a decrease of about 10%. 6 times.

[0073] 3.4 Animal efficacy study of gPT protein produced by recombinant strains of Bordetella pertussis Using reference to the component content of commercialized diphtheria, pertussis, and tetanus vaccines currently under application in China, purified gPT was adsorbed with a commercial aluminum hydroxide adjuvant and mixed with other pertussis antigen components and DT / TT antigen components at a final concentration of 25 μg / dose as the test sample. The challenge bacterium was Bordetella pertussis strain CMCC58030 (18323). Different dilutions of 80,000, 8,000, 800, 80, and 80 were used as the control group challenge solution for determination of the LD50 (LD50) of the challenge solution. The LD50 of the control group was calculated using the Reed-Muench method. Using the pertussis potency reference vaccine from the China National Institute for Food and Drug Control as the reference, the reference and test samples were diluted to high, medium, and low concentrations, respectively, according to the method for determining the potency of pertussis vaccine stock solutions in adsorbed acellular diphtheria, pertussis, and tetanus combination vaccines in the current edition of the Pharmacopoeia of the People's Republic of China. Each dilution was intraperitoneally injected into mice (half male and half female). Twenty-one days after immunization, the challenge bacterium was injected intracerebroly at each dilution. Animals were observed for 14 days after challenge. The potency of the vaccine to be tested was calculated using the mass-parallel line method, using the current software published by the China Food and Drug Inspection Institute. The potency per individual dose should be no less than 4.0 IU, and the lower limit of the 95% confidence interval should be no less than 2.0 IU. Animal experiments and statistical analysis showed that the pertussis potency of the gene-detoxified diphtheria, pertussis, and tetanus vaccine was 14.329 IU / 7.486 IU (mL), which is acceptable.

[0074] In summary, the present invention provides a recombinant strain of attenuated Bordetella pertussis. The gene-detoxified pertussis toxin expressed by the recombinant strain of the present invention is 10% less toxic than that of the wild type. 6 times, and in the mouse challenge experiment, it was proved for the first time that the genetically detoxified pertussis toxin derived from the modified CS strain of Bordetella pertussis has good protective properties, meets the relevant requirements of the "Chinese Pharmacopoeia", and can be used to prepare products for the treatment and / or detection and / or prevention of pertussis diseases, and has good application prospects.

[0075] References: [1] Wasin Buasri, Attawut Impoolsup, Chuenchit Boonchird. et al. Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelicexchange. BMC Microbiology 2012, 12:61. [2] Shrikant Thorat, et al. Development and validation of capillaryelectrophoresis sodium dodecyl sulfate (CE-SDS) method for purity analysis ofpertussis toxin, filamentous haemagglutinin and pertactin antigens. Vaccine41 (2023) 5854–5862. The nucleotide sequence and amino acid sequence involved in the present invention are as follows: The amino acid sequence of the pertussis toxin S1 subunit of the wild-type Bordetella pertussis strain CS is SEQ ID No. 1: DPPATVYRYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFYGAASSYFEYVDTYGDNAGRILAGALATYQSEYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRMAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSF The amino acid sequence of the pertussis toxin S1 subunit expressed by the recombinant Bordetella pertussis strain mS1 is SEQ ID No. 2: DPPATVYKYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFYGAASSYFEYVDTYGDNAGRI LAGALATYQSGYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRMAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSF Plasmid pJZ-G-UD nucleotide sequence information (SEQ ID No. 3): Plasmid pJZ-G-mS1 nucleotide sequence information (SEQ ID No. 4): galk The nucleotide sequence of the gene is SEQ ID No. 5: 5' homology arm (950 bp) - 3' homology arm (1650 bp) nucleotide sequence information SEQ ID No. 6: 5' homology arm (950 bp) - mS1 - 3' homology arm (1650 bp) nucleotide sequence information SEQ ID No. 7:

Claims

1. A method for constructing a recombinant strain of Bordetella pertussis, characterized in that: The construction method comprises the following steps: 1) Construction of the knockout plasmid pJZ-G-UD containing the upstream and downstream homologous recombination arms of the pertussis toxin S1 subunit gene and the knock-in plasmid pJZ-G-mS1 containing the upstream and downstream homologous recombination arms of the pertussis toxin S1 subunit gene and the pertussis toxin S1 subunit double-site mutation gene; The structure of the knockout plasmid pJZ-G-UD is: plasmid replication origin p15A ori- plasmid transfer origin oriT- sacB Promoter- sacB -Resistance genes- galk Promoter- galk -lac promoter-upstream (5') homology arm-downstream (3') homology arm, wherein the direction of the resistance gene and the plasmid replication origin p15A ori is opposite to that of other genes on the plasmid; The knock-in plasmid pJZ-G-mS1 has the structure of plasmid replication origin p15A ori- plasmid transfer origin oriT- sacB Promoter- sacB -Resistance genes- galk Promoter- Galk -lac promoter-upstream (5') homology arm-pertussis toxin S1 subunit double-site mutation gene-downstream (3') homology arm, wherein the direction of the resistance gene and the plasmid replication origin p15A ori is opposite to that of other genes on the plasmid; 2) Preparation of wild-type Bordetella pertussis competent cells; 3) Transforming the knockout plasmid pJZ-G-UD constructed in step 1) into the wild-type Bordetella pertussis competent cells obtained in step 2), resuscitating the cells, and subjecting them to forward screening and reverse screening to obtain a recombinant Bordetella pertussis strain ΔS1 lacking the pertussis toxin S1 subunit gene; 4) Preparation of competent cells of the recombinant Bordetella pertussis strain ΔS1; 5) The knock-in plasmid pJZ-G-mS1 constructed in step 1) was transformed into competent cells of the recombinant Bordetella pertussis ΔS1 strain obtained in step 4), and the cells were revived and subjected to forward screening and reverse screening to obtain a recombinant Bordetella pertussis strain with a double-site mutation in the S1 subunit.

2. The construction method according to claim 1, characterized in that: The Bordetella pertussis is the Bordetella pertussis CS strain, and the resistance gene is the gentamicin resistance gene. GmR .

3. The construction method according to claim 1, characterized in that: In step 1), the nucleotide sequence of the knockout plasmid pJZ-G-UD is SEQ ID No. 3, and the nucleotide sequence of the knockin plasmid pJZ-G-mS1 is SEQ ID No.

4.

4. The construction method according to claim 1, characterized in that: Step 3) The conversion is performed by electroporation, wherein the parameters of the electroporation are set as the distance between the electrodes of the shock cup is 0.1-5 mm, the capacitance is 15-35 μF, the resistance is 100-300 Ω, the voltage is 2000-3000 V, and the electroporation time is no more than 5.5 ms.

5. The construction method according to claim 1, characterized in that: Step 3) The forward screening comprises spreading the revived bacterial solution on a Bordet-Gengo solid medium plate containing gentamicin and culturing at 36±1°C for 5 to 7 days; the reverse screening comprises spreading the bacterial solution obtained in the forward screening on a Bordet-Gengo solid medium plate containing sucrose and 2-deoxygalactose and culturing at 36±1°C for 5 to 7 days.

6. The construction method according to claim 1, characterized in that: Step 5) The conversion is performed by electroporation, wherein the parameters of the electroporation are set as the distance between the electrodes of the shock cup is 0.1-5 mm, the capacitance is 15-35 μF, the resistance is 100-300 Ω, the voltage is 2000-3000 V, and the electroporation time does not exceed 5.5 ms.

7. The construction method according to claim 1, characterized in that: Step 5) The forward screening comprises spreading the revived bacterial solution on a Bordet-Gengo solid medium plate containing gentamicin and culturing at 36±1°C for 5 to 7 days; the reverse screening comprises spreading the bacterial solution obtained in the forward screening on a Bordet-Gengo solid medium plate containing sucrose and 2-deoxygalactose and culturing at 36±1°C for 5 to 7 days.

8. A recombinant Bordetella pertussis strain obtained according to the construction method according to any one of claims 1 to 7.

9. Use of the recombinant Bordetella pertussis strain according to claim 8 in the preparation of a product for treating and / or preventing pertussis.

10. The application according to claim 9, characterized in that: The product described is pertussis vaccine or diphtheria, pertussis and tetanus vaccine.

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