A recombinant attenuated bordetella pertussis strain and its construction method and application

Abstract

The application discloses a recombinant attenuated Bordetella pertussis strain and a construction method and application thereof, and belongs to the technical field of microbial genetic engineering. The application provides a recombinant attenuated Bordetella pertussis strain, and the gene detoxified pertussis toxin expressed by the recombinant strain is 10 6 times lower in toxicity than the wild type, and the gene detoxified pertussis toxin derived from the CS strain of Bordetella pertussis is proved to have good protectivity in a mouse challenge experiment for the first time, and can be used for preparing products for treating or / and detecting or / and preventing the pertussis disease, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial genetic engineering technology, and particularly relates to a recombinant attenuated Bordetella pertussis strain and a construction method and application thereof. BACKGROUND

[0002] Pertussis toxin (PT) is a major virulence factor produced by Bordetella pertussis, which is an A / B constitutive bacterial toxin composed of S1, S2, S3, S4, and S5 subunits in a 1:1:1:2:1 ratio. The A monomer is composed of the S1 subunit and has ADP-ribosyltransferase activity, which can block the binding of adenylate cyclase to the receptor, leading to an increase in intracellular cAMP (cyclic adenosine monophosphate) and producing various biological effects such as histamine sensitization, leukocytosis, and insulin secretion. The B oligomer is composed of S2, S3, S4, and S5 and is a non-toxic polymer that binds to various receptors on the surface of eukaryotic cells to transport the S1 subunit and exert its physiological toxicity. At the same time, the B oligomer also has the function of activating and regulating immune response.

[0003] Pertussis toxin is a key component of the currently available vaccine, and antibodies against this toxin protect children from pertussis disease. However, due to the biological toxicity of the S1 subunit, pertussis toxin must be detoxified before it can be used in vaccine preparation. Currently, the main methods for detoxifying PT are chemical detoxification and genetic detoxification. The reagents used in chemical detoxification mainly include formaldehyde, glutaraldehyde, and hydrogen peroxide. Through the modification and combination of chemical groups with the active groups of amino acids on the surface of proteins, the biological activity is removed. Due to the randomness of chemical reactions and the destruction of the multi-dimensional structure of proteins by chemical reactions, which further affects their immunogenicity, the control of chemical detoxification process has always been a difficulty in product development, which also affects the protective effect of the vaccine. Genetic detoxification targets the ADP-ribosyltransferase activity of the S1 subunit by mutating the R9K / E129G amino acids in the S1 subunit. These two site mutations significantly reduce the enzyme activity of the S1 subunit, i.e., biological toxicity, while basically retaining the structural properties of natural PT, with the surface antigenic determinants being maximally preserved, resulting in better protection. Moreover, the stability of genetic detoxification also solves the problem of process control in chemical detoxification.

[0004] Currently, the gene detoxification of PT is basically derived from the European and American pertussis Tohama strain (BAA-589), including the gene attenuated diphtheria-pertussis-tetanus vaccine which has been used for adolescents and adults. However, compared with the Tohama strain, the S1 subunit of the CS strain used for Chinese pertussis vaccine production is different at the 194th amino acid (methionine at the 194th of the CS strain and isoleucine at the 194th of the Tohama strain), and there is also a big difference between the domestic clinical epidemic strain and the foreign epidemic strain. Therefore, whether the PT with the same site mutation (such as R9K and / or E129G) also has a similar protective effect based on the gene detoxification PT of the Tohama strain is worth investigating in the real world. According to the data reported in the public, the only gene attenuated modification of the pertussis Bordetella CS strain is Wuhan Institute of Biological Products Co., Ltd., which constructed a PT gene detoxified recombinant strain by using a sequence fragment with a resistance gene through a non-scarless gene editing technology in the patent (CN 117844719 A) published in 2023. Although it is proved that the PT toxicity is reduced, there is no direct evidence to prove that it has effective protection; moreover, a new functional group sequence, kanamycin resistance sequence, is introduced into the constructed strain, which not only introduces a resistance gene into the subsequent production strain, bringing additional risks to production, but also the expression amount of the gene detoxified PT (gPT) is very low, less than 0.8 mg / L of fermentation broth, which is difficult to realize large-scale production.

[0005] Therefore, based on the pertussis Bordetella CS strain used for Chinese pertussis vaccine production, constructing a new gene detoxified strain without gene modification and with high expression will have significant scientific significance and social value, and will lay a foundation for the research and development of new pertussis vaccine. SUMMARY

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

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] The present application provides a construction method of a pertussis Bordetella recombinant strain, which comprises the following steps:

[0009] 1) constructing a knockout plasmid pJZ-G-UD containing upstream and downstream homologous recombination arms of the pertussis toxin S1 subunit gene and a knock-in plasmid pJZ-G-mS1 containing upstream and downstream homologous recombination arms of the pertussis toxin S1 subunit gene and a pertussis toxin S1 subunit double-site mutation gene;

[0010] The structure of the knockout plasmid pJZ-G-UD is plasmid replication origin p15A ori-plasmid transfer origin oriT- sacBPromoter sacB Resistance gene galk Promoter galk -lac promoter-upstream (5') homologous arm-downstream (3') homologous arm, wherein the direction of the resistance gene and the plasmid replication origin p15A ori is opposite to other genes on the plasmid;

[0011] The structure of the knock-in plasmid pJZ-G-mS1 is plasmid replication origin p15A ori-plasmid transfer origin oriT sacB Promoter sacB Resistance gene galk Promoter Galk -lac promoter-upstream (5') homologous arm-pertussis toxin S1 subunit double site mutant gene-downstream (3') homologous arm, wherein the direction of the resistance gene and the plasmid replication origin p15A ori is opposite to other genes on the plasmid;

[0012] 2) Prepare wild-type Bordetella pertussis competent cells;

[0013] 3) Transform the knock-out plasmid pJZ-G-UD constructed in step 1) into the wild-type Bordetella pertussis competent cells obtained in step 2), recover, and sequentially perform forward screening and reverse screening to obtain a recombinant Bordetella pertussis strain ΔS1 with a deleted S1 subunit gene;

[0014] 4) Prepare a recombinant Bordetella pertussis strain ΔS1 competent cell;

[0015] 5) Transform the knock-in plasmid pJZ-G-mS1 constructed in step 1) into the recombinant Bordetella pertussis strain ΔS1 competent cell obtained in step 4), recover, and sequentially perform forward screening and reverse screening to obtain a recombinant Bordetella pertussis strain with a S1 subunit double site mutation.

[0016] Further, the Bordetella pertussis is a Bordetella pertussis CS strain, and the resistance gene is a gentamicin resistance gene GmR.

[0017] Further, the nucleotide sequence of the knock-out plasmid pJZ-G-UD in step 1) is SEQ ID No. 3, and the nucleotide sequence of the knock-in plasmid pJZ-G-mS1 is SEQ ID No. 4.

[0018] Further, the preparation method of the competent cell in step 2) is inoculating the recovered wild-type Bordetella pertussis CS strain into Stainer-Scholte liquid medium and culturing to OD 550nm= 2.8~3.2, centrifugal collection of bacterial bodies, washing of bacterial bodies with 2~8℃ sterile water, then washing of bacterial bodies with 2~8℃ 10% glycerol twice, then resuspension of bacterial bodies with 2~8℃ 10% glycerol, and then obtaining.

[0019] Further, the transformation in step 3) is an electrotransformation, and parameters of the electrotransformation are set as electrode distance of 0.1~5mm, capacitance of 15~35μF, resistance of 100~300Ω, voltage of 2000~3000V, and electrotransformation time of no more than 5.5ms.

[0020] Further, the transformation in step 3) is an electrotransformation, and parameters of the electrotransformation are set as electrode distance of 0.1~5mm, capacitance of 15~35μF, resistance of 100~300Ω, voltage of 2000~3000V, and electrotransformation time of no more than 5.5ms.

[0021] Further, the forward selection in step 3) is that the recovered bacterial liquid is coated on a Bordet-Gengo solid culture medium plate containing carbon powder and gentamicin, and cultured at 36±1℃ for 5~7 days; and the reverse selection is that the bacterial liquid obtained by the forward selection is coated on a Bordet-Gengo solid culture medium plate containing carbon powder, sucrose and 2-deoxygalactose, and cultured at 36±1℃ for 5~7 days.

[0022] Further, the forward selection in step 3) is that the recovered bacterial liquid is coated on a Bordet-Gengo solid culture medium plate containing carbon powder and gentamicin, and cultured at 36±1℃ for 5~7 days; and the reverse selection is that the bacterial liquid obtained by the forward selection is coated on a Bordet-Gengo solid culture medium plate containing carbon powder, sucrose and 2-deoxygalactose, and cultured at 36±1℃ for 5~7 days.

[0023] Further, the preparation method of the competent cell in step 4) is that the recovered B. pertussis recombinant ΔS1 strain is inoculated into Stainer-Scholte liquid culture medium, cultured to OD 550nm = 2.8~3.2, centrifugal collection of bacterial bodies, washing of bacterial bodies with 2~8℃ sterile water, then washing of bacterial bodies with 2~8℃ 10% glycerol twice, then resuspension of bacterial bodies with 2~8℃ 10% glycerol, and then obtaining.

[0024] Further, the transformation in step 5) is an electrotransformation, and parameters of the electrotransformation are set as electrode distance of 0.1~5mm, capacitance of 15~35μF, resistance of 100~300Ω, voltage of 2000~3000V, and electrotransformation time of no more than 5.5ms.

[0025] Further, the parameter setting of the electric transformation in step 5) is as follows: electric shock cup electrode distance 1mm, capacitance 25μF, resistance 200Ω, voltage 2500V, and electric transformation time not more than 5ms.

[0026] Further, the forward screening in step 5) is that the recovered bacterial liquid is coated on a Bordet-Gengo solid culture medium plate containing gentamicin, and cultured at 36±1℃ for 5-7 days; and the reverse screening is that the bacterial liquid obtained by the forward screening is coated on a Bordet-Gengo solid culture medium plate containing sucrose and 2-deoxygalactose, and cultured at 36±1℃ for 5-7 days.

[0027] Further, the Bordet-Gengo solid culture medium containing gentamicin for the forward screening in step 5) is a Bordet-Gengo solid culture medium plate containing 50ppm gentamicin; and the Bordet-Gengo solid culture medium containing sucrose and 2-deoxygalactose for the reverse screening is a Bordet-Gengo solid culture medium plate containing 15% sucrose and 0.1% 2-deoxygalactose.

[0028] The Bordet-Gengo solid culture medium plate containing 15% sucrose and 0.1% 2-deoxygalactose (DOG) for the reverse screening is obtained by a large number of screening of the present application. Under the combined action of 15% sucrose and 0.1% DOG, the secondary homologous recombination rate is significantly improved, which is increased from 20% under the condition of 0.1% DOG alone and less than 10% under the condition of 15% sucrose alone to more than 95%. In the case of ensuring a high recombination rate, the single use concentration of DOG is effectively reduced.

[0029] The present application also provides a recombinant Bordetella pertussis strain obtained by the above construction method.

[0030] The present application also provides an application of the above recombinant Bordetella pertussis strain in preparing a product for treating and / or preventing pertussis disease.

[0031] Further, the product is a pertussis vaccine or a DPT vaccine.

[0032] The present application has the following beneficial effects: 1) In the present application, the recombinant Bordetella pertussis strain is constructed by using electroporation to introduce a suicide plasmid carrying the target nucleotide sequence, so as to complete the editing and modification of the target gene. Compared with the traditional method of using E. coli to introduce a recombinant plasmid, the nucleic acid introduction method in the present application avoids the introduction and pollution of E. coli, and 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 subculture of the strain, and will not pollute the genetic background. 2) The recombinant Bordetella pertussis strain prepared in the present application, whether it is a S1 subunit double-site mutant (R9K / E129G) strain or a S1 subunit full-length deletion strain, will not introduce resistance genes, other functional genes and additional nucleotide sequences into the genome, and will not increase the risk of strain stability, biological safety and subsequent production; 3) The gene detoxified PT expressed by the S1 subunit double-site mutant recombinant strain prepared in the present application has a toxicity reduction of 10 6 times compared with the WHO second-generation pertussis toxin standard 15 / 126; and in the mouse challenge experiment, it is first proved that the gPT derived from the genetic modification of Bordetella pertussis CS strain has good protective effect, which meets the requirements of the Chinese Pharmacopoeia; 4) The gPT expression amount of the S1 subunit double-site mutant recombinant strain prepared in the present application is consistent with the expression amount of the recombinant strain derived from Bordetella pertussis Tohama strain (BAA-589) of Bionet company reported in the literature, which meets the demand of large-scale production. The present application lays a foundation for the study of the mechanism of PT protein of Bordetella pertussis CS strain and the study of gene detoxified pertussis vaccine.

[0033] In summary, the present application provides a attenuated Bordetella pertussis recombinant strain, the gene detoxified pertussis toxin expressed by the recombinant strain of the present application has a toxicity reduction of 10 6 times compared with the wild type, and in the mouse challenge experiment, it is first proved that the gene detoxified pertussis toxin derived from the genetic modification of Bordetella pertussis CS strain has good protective effect, which meets the requirements of the Chinese Pharmacopoeia, and can be used for preparing products for treating or / and detecting or / and preventing pertussis disease, and has good application prospect.

[0034] Definitions of terms used in the present application: unless otherwise specified, the initial definition of the term provided herein is applicable to the term throughout the specification; for the terms not specifically defined herein, the meanings should be given to the skilled person according to the disclosure and the context.

[0035] The term "scarless gene editing" refers to the modification of a target gene without introducing additional DNA sequences, usually by homologous recombination: using the upstream and downstream homology arms of a specific site flanking the target gene to be edited, introducing the nucleotide sequence of the target gene and the screening gene nucleotide sequence in the genome by forward screening; obtaining the secondary recombination strain that deletes the screening gene nucleotide sequence and retains the target gene nucleotide sequence by reverse screening. Scarless editing can realize the superposition of multiple gene modifications. Unlike scarless gene editing, for example, using resistance substitution method for scarred gene editing, the modification is successful while introducing resistance genes.

[0036] The term "homology arm" refers to the flanking sequence upstream and / or downstream of a specific site to be inserted or replaced by a target gene, or a sequence having at least 95% or more sequence identity with the flanking sequence. The length of the homology arm can generally be several hundred bp, even more than 1000 bp.

[0037] The term "forward screening" refers to identifying the integration of an exogenous sequence in the Bordetella pertussis genome by forward screening the presence and / or expression of a gene.

[0038] The term "reverse screening" refers to identifying the deletion of a screening nucleotide sequence from the Bordetella pertussis genome by reverse screening the absence and / or non-expression of a gene.

[0039] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common practice in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present application.

[0040] The above content of the present application will be further described in detail through the following specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 The structure of pJZ-G-UD plasmid and pJZ-G-mS1 plasmid is shown.

[0042] Figure 2 The PCR verification results of the primary recombination strain grown by pJZ-G-UD transformation are shown. Wherein, A is sacB primer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0043] Figure 3 The PCR verification results of the secondary recombination strain grown by pJZ-G-UD transformation are shown. Wherein, A is sacBPrimer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0044] Figure 4 PCR verification results of a secondary recombination strain grown by pJZ-G-mS1 transformation. Wherein, A is sacB Primer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0045] Figure 5 PCR verification results of a secondary recombination strain grown by pJZ-G-mS1 transformation. Wherein, A is sacB Primer verification, B is S1 primer verification, C is upstream homologous recombination primer verification, and D is downstream homologous recombination primer verification.

[0046] Figure 6 Growth curves of the recombinant strain mS1 and the wild type Bordetella pertussis CS strain (WT).

[0047] Figure 7 PT protein time curve of the recombinant strain mS1 and the wild type Bordetella pertussis CS strain (WT).

[0048] Figure 8 Comparison electrophoregram of wild type pertussis toxin (red) and genetically detoxified pertussis toxin (blue).

[0049] Figure 9 CHO cell determination map of different cluster degrees. DETAILED DESCRIPTION

[0050] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0051] In the following experiments, if the temperature is not specified, it is a normal temperature reaction, and the normal temperature is room temperature, which is 25±5℃.

[0052] The preservation number of the Bordetella pertussis CS strain used in the following examples is CMCC 58003. CHO cells (ATCC-CCL-61) are provided by the Product Technology Room of Chengdu Institute of Biological Products Co., Ltd. Stainer-Scholte liquid medium (SSM) and carbon Bordet-Gengou (BG) medium are prepared and provided by the Medium Room of Chengdu Institute of Biological Products Co., Ltd. CHO cell culture medium DMEM / F12 (1:1) (item number: 11330032) is purchased from Gibco Company. Mice for efficacy experiments are provided by the Animal Room of Chengdu Institute of Biological Products Co., Ltd.

[0053] R9K represents that the arginine (Arginine, abbreviated as R) at the 9th position of the S1 subunit of the pertussis toxin is mutated to lysine (Lysine, abbreviated as K), and E129G represents that the glutamic acid (Glutamic acid, abbreviated as E) at the 129th position of the S1 subunit of the pertussis toxin is mutated to glycine (Glycine, abbreviated as G).

[0054] Example 1: Preparation method of attenuated Bordetella pertussis recombinant strain

[0055] I. Preparation method of PT-S1 subunit deletion Bordetella pertussis recombinant strain

[0056] 1.1 Construction of plasmids pJZ-G-UD and pJZ-G-mS1

[0057] By means of gene synthesis and enzyme digestion and ligation, the sequence (with a promoter) is inserted on the suicide plasmid with gentamicin resistance, sacB gene function, named pJZ-G; galk The sequence (with a promoter) is inserted on the suicide plasmid with gentamicin resistance, sacB and galk gene function, named pJZ-G; galk The nucleotide sequence information of the gene is shown in SEQ ID No. 5:

[0058] The plasmid pJZ-G-UD is obtained by integrating the 5' homologous arm (950bp)-3' homologous arm (1650bp) of the PT-S1 subunit gene into the plasmid pJZ-G by gene synthesis, taking the restriction enzyme digestion sites SacI and BamHI in the multiple cloning region of the plasmid as the fragment connection sites; the nucleotide sequence information of the 5' homologous arm (950bp)-3' homologous arm (1650bp) is shown in SEQ ID No. 6:

[0059] The plasmid pJZ-G-UD contains ori-p15A, gentamicin resistance gene ( GmR ), sacB and galk anti-screening gene, and the multiple cloning site region contains 5' homologous arm and 3' homologous arm, and its structure is shown in Figure 1 .

[0060] The plasmid pJZ-G-UD is used to realize the knockout of the target gene S1 subunit to be edited.

[0061] The plasmid pJZ-G-mS1 is obtained by inserting the nucleotide sequence of the target gene (mS1) into the upstream (5') homologous arm (950 bp) and the downstream (3') homologous arm (1650 bp) in the multiple cloning region of the plasmid pJZ-G-UD by gene synthesis and enzyme digestion and ligation. In theory, 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 the mutation of two specific sites of the pertussis toxin S1 subunit. The nucleotide sequence information of the 5' homologous arm (950 bp)-mS1-3' homologous arm (1650 bp) is shown in SEQ ID No. 7. The plasmid pJZ-G-mS1 contains ori-p15A, a gentamicin resistance gene (GmR), a counter-selection gene, and a multiple cloning site region containing a 5' homologous arm-mS1-3' homologous arm, and its structure is shown in SEQ ID No. 8. GmR 、 sacB and galk a counter-selection gene. The multiple cloning site region contains a 5' homologous arm-mS1-3' homologous arm, and its structure is shown in SEQ ID No. 8. Figure 1

[0062] Using the plasmid pJZ-G-UD and the plasmid pJZ-G-mS1, the double-site mutation of the S1 subunit is realized by the knockout of the target gene to be edited and the knockin of the target gene, respectively.

[0063] 1.2 Preparation of competent cells

[0064] The wild-type Bordetella pertussis CS strain bacteria stored at -80°C were taken out of the refrigerator, quickly thawed in a biological safety cabinet, inoculated into 5 mL of SSM medium, and cultured at 36±1°C, 250 rpm for 24 hours.

[0065] The recovered bacterial liquid was inoculated into 50 mL of SSM medium, and the final concentration of the inoculated bacteria was adjusted to about 200 million CFU / mL. The culture was incubated at 36±1°C, 250 rpm for 22-24 hours, and the OD 550 was about 3.0.

[0066] In a biological safety cabinet, the bacterial liquid was collected in a 50 mL sterile centrifuge tube, centrifuged at 4000xg at 4°C for 15 minutes, and the bacterial cells were collected. g The bacterial cells were resuspended in 25 mL of ice water bath-precooled sterile double distilled water, washed by repeatedly gently blowing with a pipette and a sterile pipette, and then centrifuged at 4000xg at 4°C for 15 minutes to collect the bacterial cells again.

[0067] g The bacterial cells were resuspended in 10 mL of ice water bath-precooled 10% (V / V) glycerol, quickly and gently dispersed with a pipette and a sterile pipette, and then placed in ice water for 10 minutes. The bacterial cells were collected again by centrifugation at 4000xg at 4°C for 15 minutes.

[0068] The bacterial cells were resuspended in 10 mL of ice water bath-precooled 10% (V / V) glycerol, quickly and gently dispersed with a pipette and a sterile pipette, and then placed in ice water for 10 minutes. The bacterial cells were collected again by centrifugation at 4000xg at 4°C for 15 minutes. g ​​Centrifuge for 15 minutes under the condition, collect the bacteria, and repeat the operation twice;

[0069] After resuspending the bacteria in 1 mL ice water bath pre-cooled 10% (V / V) glycerol and mixing, divide 100 μL per tube, and store at -80℃ for standby or directly transfer to the electroporation cup pre-cooled in ice water bath for preparation of electroporation.

[0070] 1.3 Electroporation and plate screening

[0071] Take 100 μL competent cells prepared in step 1.2 from -80℃ refrigerator, thaw on ice, and place the electroporation instrument shock cup (1 mm interval) and the plasmid pJZ-G-UD prepared in step 1.1 on ice for pre-cooling for 5 min;

[0072] In the biological safety cabinet, add 1 μg plasmid pJZ-G-UD to 100 μL competent cells, mix, and then transfer to the electroporation cup, and operate in ice water bath all the time;

[0073] Set the parameters: 2500 V, 25 μF, 200 Ω, ensure that the bacteria and plasmid mixture is at the bottom of the shock cup, wipe the condensate outside the shock tank, place in the shock cup, and start the electric pulse;

[0074] After the shock is completed, take out the sample pool as soon as possible, immediately add 1 mL pre-heated at 37℃ SSM medium, transfer to a sterile centrifuge tube, and recover at 36±1℃, 250 rpm for 12 h on a constant temperature shaker;

[0075] Spread the bacterial liquid on a BG resistance plate containing 50 ppm gentamicin, and culture at 36±1℃ for 5-7 days, and select the strain with positive gentamicin resistance.

[0076] 1.4 Recombinant strain gene level detection

[0077] Use a sterile gun head to pick a single colony growing on the gentamicin resistance screening plate, streak on a new gentamicin resistance BG plate, and dip the remaining bacteria on the gun head in 20 uL sterile water as a PCR verification template; use plasmid sacB primers and S1 internal primers to verify whether homologous recombination between the plasmid and the bacterial genome occurs; further use up-end homologous arm integration specific primers and down-end homologous arm integration specific primers to verify whether the plasmid is integrated by single exchange of the upstream homologous arm or single exchange of the downstream homologous arm. The primers used in PCR verification are shown in Table 1.

[0078] Table 1 Knockout recombination verification primer table

[0079]

[0080] The PCR verification results of a recombinant strain transformed from pJZ-G-UD are shown in Figure 2 Figure 2 A uses primers Text Sacb s and Text Sacb as to randomly select 1 # -8 # strain, 951 bp fragments are cloned from all strains sacB sequences, and Figure 2 B uses primers Text S1wb s and Text S1wb as to randomly select 1 # -8 # strains, 319 bp and 1129 bp fragments are cloned from all strains, indicating that 1 # -8 # The cloned strains all have single exchange of homologous arms, and the sequences on the plasmid are integrated into the genome; Figure 2 C uses homologous arm integration specific primers Text up s and Text up as to clone 1150 bp fragments, Figure 2 D uses homologous arm integration specific primers to clone 2660 bp fragments, and the above results prove that 1 # -8 # The strains all have single exchange recombination through the upstream homologous arm. The first homologous recombination of the Bordetella pertussis recombinant strain is obtained, and is named UP-sacB + -galk + 1 # -8 # Lane 9: template is water, lane 10: template is pJZ-G-UD plasmid, lane 11: template is pJZ-G-mS1 plasmid, and lane 12: template is wild-type Bordetella pertussis strain.

[0081] 1.5 plate screening 2

[0082] Randomly select one strain UP-sacB + -galk + After the strain is amplified by SSM medium, different dilution multiples of the bacterial liquid are added or streaked on BG medium with sucrose (15%) and DOG (0.1%) concentrations, and cultured at 36±1℃ for 5-7 days;

[0083] If the upstream or downstream homologous recombination arm of the S1 subunit does not have second homologous recombination, under the action of the gene sacB + -galk + The bacteria will metabolize sucrose and DOG, have a lethal effect, and will not grow on the BG plate; if the homologous recombination arm has second homologous recombination, the genome is lost​sacB + -galk + After gene generation, bacteria will be able to grow on BG plates.

[0084] 1.6 Gene-level detection of recombinant strains 2

[0085] Following the procedure in 1.5, the single clones grown on the reverse screening plate were verified by PCR, and the results are as follows. Figure 3 Middle 1 # -18 # The results of the strains are shown. Lanes 1-14 are single clones grown on sucrose / DOG carbon powder solid plates, lane 15 is templated with water, lane 16 is templated with pJZ-G-UD plasmid, lane 17 is templated with pJZ-G-mS1 plasmid, and lane 18 is templated with wild-type pertussis strain; Figure 3 A in 1 # -14 # The strain did not amplify a band using primers Text Sacb s and Text Sacb as, indicating that a second homologous recombination occurred under the action of sucrose and DOG reverse screening genes, successfully losing the sequence in the non-upstream and downstream homologous recombination arm region of the plasmid. Figure 3 In B, primers Text S1wb s and Text S1wb as are used for lane 1. # 3 # 4 # 6 # 7 # 8 # 10 # and 13 # Only 319bp was amplified, and then... Figure 3 PCR results using up- and down-specific primers for the corresponding monoclonal strains of C / D indicate that the second homologous recombination occurred in the downstream homologous recombination arm, successfully obtaining a recombinant strain of Bordetella pertussis with the PT S1 subunit deletion, 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 reverted to its wild-type state. Lane 11... # 12 # and 14 # The simultaneous amplification of 319bp and 1129bp indicates that the selected monoclonal strain is impure and that there are strains that have undergone a second homologous recombination upstream and downstream.

[0086] 1.7 Preservation of Recombinant Strains

[0087] The verified monoclonal strain ΔS1 is coated on a carbon BG solid plate and grown at 36±1°C for 5-7 days; the bacteria are scraped off the plate and transferred to SSM medium, which is cultured at 36±1°C and 250 rpm for 24-28 hours, with an OD value of about 4-5; a 40% glycerol solution is prepared and autoclaved; the bacterial solution is mixed with the 40% glycerol solution at a ratio of 1:1, evenly mixed, and then distributed into sterile cell freezing tubes and stored in a-80°C refrigerator.

[0088] II. Preparation method of a recombinant Bordetella pertussis strain with PT-S1 subunit double-site mutation (R9K / E129G)

[0089] 2.1 Construction of knock-in plasmid pJZ-G-mS1

[0090] The 5' homologous arm (950 bp) of the PT-S1 subunit gene, the S1 subunit double-site mutation gene, and the 3' homologous arm (1650 bp) of the PT-S1 subunit gene are integrated into the pJZ-G plasmid by gene synthesis to obtain the plasmid pJZ-G-mS1, with the 5' homologous arm (950 bp)-mS1-3' homologous arm (1650 bp) nucleotide sequence information being SEQ ID No. 7:

[0091] The plasmid pJZ-G-mS1 contains ori-p15A, a gentamicin resistance gene (GmR), a sacB and galk a counter-screening gene, and a multiple cloning site region containing a 5' homologous arm, an S1 subunit double-site mutation (R9K / E129G) sequence, and a 3' homologous arm, which has the structure as shown in Figure 1 .

[0092] 2.2 Preparation of competent cells

[0093] The PT-S1 subunit-deleted Bordetella pertussis ΔS1 strain is taken out from the-80°C refrigerator, and the preparation of competent cells is performed according to step 1.2.

[0094] 2.3 Electroporation and plate screening 1

[0095] The competent cells prepared in step 2.2 are subjected to electroporation according to the procedure of step 1.3, and the bacterial solution is coated on a carbon BG resistance plate containing 50 ppm gentamicin and cultured at 36±1°C for 5-7 days, to screen out strains with positive gentamicin resistance.

[0096] 2.4 Recombinant strain gene level detection 1

[0097] Following the procedure in 1.4, the single clones grown on the positive screening plate were verified by PCR. The primers used for PCR verification are shown in Table 2.

[0098] Table 2 Primer list for validation of S1 subunit dual-site mutation (mS1) knock-in recombination.

[0099]

[0100] The PCR verification results of the recombinant strain grown from pJZ-G-mS1 transformation are as follows: Figure 4 As shown, Figure 4 A uses primers Text Sacb s, Text Sacb as is randomly selected 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 # The simultaneous cloning of two fragments, 319 bp and 1129 bp, in the strain indicates that 1 # -6 # All cloned bacteria underwent single crossover of homologous 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 1960bp fragment was cloned from the strain, indicating that strain #5 underwent its first homologous recombination in the upstream homologous recombination arm; Figure 4 D uses primers Text down s, Text down as in 1 # 2 # 3 # 4 # and 6 # A 2660bp fragment was cloned from the strain, indicating that 1 # 2 # 3 # 4 # and 6 # The strain underwent its first homologous recombination in the downstream homologous recombination arm. The Bordetella pertussis ΔS1 strain resulting from this first homologous recombination was named... UP- or DOWN-sacB + -galk + ΔS1 1 # -6 #Lane 1-6 PCR template is single clone grown on GM50ppm carbon end solid plate, lane 7 template is water, lane 8 template is pJZ-G-UD plasmid, lane 9 template is pJZ-G-mS1 plasmid, lane 10 template is wild type pertussis strain.

[0101] 2.5 Plate screening 2

[0102] Any one of each selected strain UP-sacB + -galk + ΔS1 Strain and DOWN-sacB + -galk + ΔS1 After the strain is amplified by SSM medium, it is re-streaked and coated on carbon end BG medium with sucrose (15%)+DOG (0.1%) concentration, and cultured at 36±1℃ for 5-7 days.

[0103] 2.6 Recombinant strain gene level detection 2

[0104] According to the operation process of 2.5, the single clone grown on the reverse screening plate is verified by PCR, and the results are shown in Figure 5 .Among them UP-sacB + -galk + ΔS1 6 single clones (A lanes 1-6) are selected from the bacteria; Figure 5 DOWN-sacB + -galk + ΔS1 14 single clones (A lanes 7-20) are selected from the bacteria. Figure 5 Figure 5 1-20 # strains in A # The primer Text Sacb s and Text Sacb as are not amplified, indicating that the second homologous recombination occurs under the action of sucrose and DOG reverse screening gene, and the sequence of the non-upstream and downstream homologous recombination arm region on the plasmid is successfully lost; Figure 5 1-6 # strains in B # The first homologous recombination occurs in the upstream homologous recombination arm, and the primers Text S1wb s and Text S1wb as are used to amplify lanes 1 # , 2 # , 3 # and 5 # ​PCR products of 1129 bp were amplified from lanes 8 # and 6 # PCR products of 319 bp were amplified from lanes 7 # -20 # The first homologous recombination of the strain occurred in the downstream homologous recombination arm, and primers Text S1wb s and Text S1wb as were used to amplify lanes 7 # , 9 # , 11 # , 13 # , 17 # and 20 # PCR products of 1129 bp were amplified from lanes 8 # , 10 # , 12 # , 14 # , 15 # , 16 # and 19 # PCR products of 319 bp were amplified from lanes 7 Figure 5 C-D, primers Text up s / Text up as and Text down s / Text down as were used to amplify lanes 1 # -6 # and 7 # -20 # The difference in the molecular weight of the cloned fragments of the strains also proved that the mS1 sequence insertion sites in lanes 1 # , 2 # , 3 # , 5 # , 7 # , 9 # , 11 # , 13 # , 17 # and 20 # were between the upstream and downstream homologous arms. Finally, the PT S1 subunit double-site mutant (R9K / E129G) recombinant Bordetella pertussis strain was successfully obtained, and was named mS1. The PCR templates of lanes 1-20 were single colonies grown on sucrose / DOG carbon end solid plates, the template of lane 21 was water, the template of lane 22 was the pJZ-G-UD plasmid, the template of lane 23 was the pJZ-G-mS1 plasmid, and the template of lane 24 was the wild-type Bordetella pertussis strain;

[0105] 2.7 Preservation of recombinant strain mS1

[0106] The verified monoclonal strain mS1 was coated on a carbon BG solid plate and grown at 36±1°C for 5-7 days; the bacteria were scraped off the plate and transferred into SSM medium and cultured at 36±1°C and 250 rpm for 24-28 hours, with an OD value of about 4-5; a 40% glycerol solution was prepared and autoclaved; the bacterial solution was mixed with the 40% glycerol solution at a ratio of 1:1, uniformly mixed, and then distributed into sterile cell freezing tubes and stored in a -80°C refrigerator.

[0107] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0108] Experimental Example 1: Growth curve, gPT expression, cytotoxicity and protective efficacy of recombinant strain mS1

[0109] 1.1 Growth curve of recombinant strain mS1 and expression curve of detoxified PT protein (gPT)

[0110] Randomly selected different numbered recombinant Bordetella pertussis strain mS1 and wild type CS strain were inoculated into 50 mL SSM medium in a 250 mL flask, with a final concentration of 200 million CFU / mL. The flask was placed in a constant temperature shaker at 36±1°C and 250 rpm, and the OD value of the bacterial solution was detected at 0h, 16h, 19h, 22h, 25h and 28h, respectively. 550 The results are shown in Figure 6 The growth rates of each mS1 recombinant strain and wild type pertussis CS strain were similar.

[0111] The bacterial samples were collected at 0h, 16h, 19h, 22h, 25h and 28h, respectively, and centrifuged at room temperature and 12000 rpm for 8 minutes to collect the culture supernatant. The samples were diluted 50-fold and 100-fold, respectively, and detected by PT monoclonal antibody double sandwich ELISA method. The standard curve of PT protein detection range of 1.5625-200 ng / mL was drawn by diluting the purified wild type PT antigen by multiple times, and the corresponding values of the samples within the range were multiplied by the dilution factor to obtain the arithmetic mean, and the gPT content in the collected culture supernatant was obtained. The results of the time-effect curve are shown in Figure 7 The gPT protein content secreted by the recombinant Bordetella pertussis strain increased with the increase of culture time, with a maximum value of about 2800-3000 ng / mL, which was basically consistent with the gPT expression amount of the strain based on the gene modification of pertussis Tohama strain reported in the literature [1].

[0112] 3.2 Analysis of the structural integrity of PT protein expressed by recombinant strain mS1 by capillary electrophoresis (CE)

[0113] The supernatant of the recombinant strain mS1 and wild type strain cultured for 28 hours in step 3.1 was used to purify gPT and PT by column chromatography, respectively, and the amount of protein was quantified by Lowry method and each subunit of the protein was analyzed by capillary electrophoresis.

[0114] Not less than 200 μg of each of the gPT and PT samples to be tested was concentrated to 2 mg / mL by using 10 kD ultrafiltration centrifuge tubes. The sample was then exchanged twice with sample buffer containing 100 mM Tris and 1% SDS. The concentration of the sample was maintained at 2 mg / mL. After reduction by adding a certain proportion of β-mercaptoethanol in a fume hood, the sample was transferred to a sample bottle for loading. The capillary electrophoresis conditions were as follows: sample loading voltage -10 kV, migration voltage -15 kV; detection wavelength 220 nm, reference wavelength 360 nm.

[0115] The results are shown in Table 3. Figure 8 As shown in Table 3, both gPT and PT protein had five independent peaks, which were designated as S1-S5 according to the time of peak appearance, which was consistent with the characteristic peak shape of pertussis toxin in the literature [2]. The peak appearance time of each subunit of gPT and PT was basically consistent.

[0116] 3.3 Detection of the cytotoxicity of PT protein of recombinant strain mS1

[0117] The in vitro toxicity of PT protein of recombinant strain mS1-8 was evaluated by CHO cell clustering experiment.

[0118] CHO cells that had grown into a dense monolayer were trypsinized and then blown into single suspension by using culture medium, and the concentration was diluted to 2 x 10 4 The WHO 2nd generation pertussis toxin standard 15 / 126 was diluted to one activity unit (1 IU / ml) as a standard, the cell culture medium was used as a negative control, the wild type pertussis toxin PT purified by column chromatography was used as a positive control, and different batches of gPT obtained by purifying the supernatant of different batches of recombinant strain mS1-8 cultured in a fermenter by column chromatography were used as samples to be tested. In the dilution plate, 125 μL of cell culture medium was used as a diluent, 250 μL of each of the above samples was added to the first column of wells, and after dilution by gradient, all were transferred to the cell plate pre-incubated with adherent cells. After 45-48 h of incubation at 37°C, the 96-well plate was observed under a microscope to observe the cell clustering. If no cell clustering occurred, it was judged as “-”, if 50% or less of the cells clustered, it was judged as “±”, if 50% or more of the cells clustered, it was judged as “+”, and if 100% of the cells clustered, it was judged as “++”. The cell clustering diagram is shown in Figure 2. Figure 9The sample toxicity results were calculated according to the concentration of the standard sample 15 / 126 final cluster hole dilution multiple of each 96-hole plate, and the calculation formula was: sample activity (IU / mg) = international standard activity (IU / mL) / sample cluster final concentration (mg / mL). The gPT cytotoxicity test results are shown in Table 3. Activity result 1 is the first round of testing, and activity result 2 is the second round of testing. gPT (batch number 20250102) did not participate in the first round of testing.

[0119] Table 3 Cytotoxicity test results of different batches of gPT

[0120]

[0121] Therefore, the in vitro toxicity of the gPT protein expressed by the different batches of recombinant strains is reduced to 0.0001% relative to wild-type pertussis toxin PT, and the toxicity is reduced by about 10 6 times.

[0122] 3.4 Animal efficacy experiment of gPT protein of Bordetella pertussis recombinant strain

[0123] According to the content of each component of the foreign commercial component DPT vaccine and the component DPT vaccine currently in the declaration stage in China, after purification, the gPT was adsorbed with the commercial aluminum hydroxide adjuvant, and the final concentration was 25 μg / dose, which was mixed with other antigen components of pertussis and DT / TT antigen components in proportion as the test product; the attack bacteria of pertussis were Bordetella pertussis CMCC58030 (18323) strain: 80000, 8000, 800, 80, and 8 different dilutions of bacterial liquid were used as the control group of attack bacteria liquid for determining the LD50 of the attack bacteria liquid. The LD50 of the control group was calculated by the Reed-Muench method; according to the method for determining the titer of the pertussis vaccine stock solution in the adsorbed acellular DPT combined vaccine in the “People's Republic of China Pharmacopoeia” (current edition), the reference product of the pertussis efficacy of the China Food and Drug Inspection Institute was used as a reference, and the reference product and the test product were diluted in high, medium and low concentrations. Each dilution was intraperitoneally injected into immune mice (half male and half female). Twenty-one days after the animal immunization, the attack bacteria were injected into the brain cavity of the mice at each dilution. The animals were observed for 14 days after the attack. The titer of the vaccine to be tested was calculated by the parallel line method, and the calculation software was the current software issued by the China Food and Drug Inspection Institute. The immune titer of each human dose should be not less than 4.0 IU, and the lower limit of the 95% confidence interval should be not less than 2.0 IU. Through animal experiments and statistical analysis, the pertussis efficacy of the genetically detoxified component DPT vaccine was 14.329 IU / 7.486 IU (mL), and the efficacy was qualified.

[0124] In summary, the present application provides a recombinant strain of attenuated Bordetella pertussis, and the genetically detoxified pertussis toxin expressed by the recombinant strain of the present application has a toxicity reduction of 10 6The prepared detoxified pertussis toxin has good protective effect in the mouse challenge experiment, and the detoxified pertussis toxin derived from the modified gene of Bordetella pertussis CS strain has good protection, meets the relevant requirements of Chinese Pharmacopoeia, and can be used for preparing products for treating or / and detecting or / and preventing pertussis disease, and has good application prospect.

[0125] Reference:

[0126] [1] Wasin Buasri, Attawut Impoolsup, Chuenchit Boonchird. et al.Construction of Bordetella pertussis strains with enhanced production ofgenetically-inactivated Pertussis Toxin and Pertactin by unmarked allelicexchange. BMC Microbiology 2012, 12:61.

[0127] [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. Vaccine 41 (2023) 5854-5862.

[0128] The nucleotide sequence and the amino acid sequence involved in the application are as follows:

[0129] Amino acid sequence of the S1 subunit of the pertussis toxin of the wild-type Bordetella pertussis CS strain SEQ ID No. 1 : DPPATVYRYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFYGAASSYFEYVDTYGDNAGRILAGALATYQSEYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRMAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSF

[0130] Amino acid sequence of the S1 subunit of the pertussis toxin expressed by the recombinant Bordetella pertussis strain mS1 SEQ ID No. 2:

[0131] DPPATVYKYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFYGAASSYFEYVDTYGDNAGRILAGALATYQSGYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRMAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSF

[0132] Nucleotide sequence information of the plasmid pJZ-G-UD (SEQ ID No. 3):

[0133]

[0134] Plasmid pJZ-G-mS1 nucleotide sequence information (SEQ ID No. 4):

[0135]

[0136] galk Nucleotide sequence of the gene SEQ ID No. 5:

[0137]

[0138] 5' homology arm (950 bp) - 3' homology arm (1650 bp) nucleotide sequence information SEQ ID No. 6:

[0139]

[0140] 5' homology arm (950 bp) - mS1 - 3' homology arm (1650 bp) nucleotide sequence information SEQ ID No. 7:

[0141]

Claims

1. A method for constructing a recombinant strain of Bordetella pertussis CS strain, characterized in that, The construction method comprises the following steps: 1) constructing a knockout plasmid pJZ-G-UD containing upstream and downstream homologous recombination arms of the S1 subunit gene of pertussis toxin and a knock-in plasmid pJZ-G-mS1 containing upstream and downstream homologous recombination arms of the S1 subunit gene of pertussis toxin and a S1 subunit double-site mutant gene of pertussis toxin; The structure of the knockout plasmid pJZ-G-UD is plasmid replication origin p15A ori-plasmid transfer origin oriT sacB promoter sacB -resistance gene 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 other genes on the plasmid; The structure of the knock-in plasmid pJZ-G-mS1 is plasmid replication origin p15A ori-plasmid transfer origin oriT sacB promoter sacB -resistance gene galk promoter Galk -lac promoter-upstream (5') homologous arm-pertussis toxin S1 subunit double site mutation gene-downstream (3') homologous arm, wherein the direction of the resistance gene and the plasmid replication origin p15A ori is opposite to other genes on the plasmid; The S1 subunit double-site mutant gene is R9K / E129G; The resistance gene is a gentamicin resistance gene GmR; The nucleotide sequence of the knockout plasmid pJZ-G-UD is SEQ ID No. 3, and the nucleotide sequence of the knock-in plasmid pJZ-G-mS1 is SEQ ID No. 4; 2) preparing a wild-type B. pertussis CS strain competent cell; 3) transforming the knockout plasmid pJZ-G-UD constructed in step 1) into the wild-type B. pertussis CS strain competent cell obtained in step 2), recovering, and sequentially performing forward screening and reverse screening to obtain a B. pertussis CS strain recombinant strain ΔS1 strain with a S1 subunit gene of pertussis toxin deleted; 4) preparing a B. pertussis CS strain recombinant strain ΔS1 strain competent cell; 5) transforming the knock-in plasmid pJZ-G-mS1 constructed in step 1) into the B. pertussis CS strain recombinant strain ΔS1 strain competent cell obtained in step 4), recovering, and sequentially performing forward screening and reverse screening to obtain a B. pertussis CS strain recombinant strain with a S1 subunit double-site mutation.

2. The method of claim 1, wherein, The transformation in step 3) is electroporation, and the parameters of the electroporation are set as follows: an electrode distance of 0.1-5 mm, a capacitance of 15-35 μF, a resistance of 100-300 Ω, a voltage of 2000-3000 V, and an electroporation time of not more than 5.5 ms.

3. The method of claim 1, wherein The forward screening in step 3) is that the recovered bacterial liquid is coated on a Bordet-Gengo solid culture medium plate containing carbon powder and gentamicin, and cultured at 36±1℃ for 5-7 days; and the reverse screening is that the bacterial liquid obtained by the forward screening is coated on a Bordet-Gengo solid culture medium plate containing carbon powder, sucrose and 2-deoxygalactose, and cultured at 36±1℃ for 5-7 days.

4. The method of claim 1, wherein, The transformation in step 5) is electroporation, and the parameters of the electroporation are set as follows: an electrode distance of 0.1-5 mm, a capacitance of 15-35 μF, a resistance of 100-300 Ω, a voltage of 2000-3000 V, and an electroporation time of not more than 5.5 ms.

5. The method of claim 1, wherein, The forward screening in step 5) is that the recovered bacterial liquid is coated on a Bordet-Gengo solid culture medium plate containing carbon powder and gentamicin, and cultured at 36±1℃ for 5-7 days; and the reverse screening is that the bacterial liquid obtained by the forward screening is coated on a Bordet-Gengo solid culture medium plate containing carbon powder, sucrose and 2-deoxygalactose, and cultured at 36±1℃ for 5-7 days.

6. The B. pertussis CS strain recombinant strain obtained by the construction method in any one of claims 1-5.

7. The use of the B. pertussis CS strain recombinant strain in claim 6 in the preparation of a product for treating and / or preventing pertussis disease.

8. Use according to claim 7, characterized in that, The product is a pertussis vaccine or a diphtheria-tetanus-pertussis vaccine. The product is a pertussis vaccine or a diphther

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