Attenuated pertussis CS recombinant bacterium as well as construction method and application thereof

Abstract

The invention provides an attenuated pertussis CS recombinant bacterium, the CS recombinant bacterium is obtained by mutating a ninth arginine residue of an S1 subunit in a CS strain with a preservation number of CMCC 58003 into a lysine residue and mutating a 129th glutamic acid residue into a glycine residue, and the amino acid sequence of the S1 subunit of the CS recombinant bacterium is as shown in SEQ ID NO.1. The invention further provides a preparation method of the attenuated pertussis CS recombinant bacterium. The method for constructing the recombinant bacteria comprises the following steps: constructing recombinant plasmids; preparing competent cells of the wild pertussis CS strain; electrically transferring the recombinant plasmid into competent cells of the wild pertussis CS strain; respectively preparing BG sheep blood screening plates containing Amp and DOG; sequentially screening a first round and a second round of recombinant pertussis CS strains; and preparing the two-round recombinant pertussis CS strain into a competent strain, and screening twice according to the same steps to obtain the attenuated pertussis CS recombinant strain. The constructed recombinant bacteria can efficiently obtain detoxified PT, and can be applied to products for treating, detecting and preventing pertussis diseases.

Description

Technical Field

[0001] This invention belongs to the field of pertussis toxin gene genetic detoxification technology, specifically relating to an attenuated pertussis CS recombinant bacterium, its construction method, and its application. Background Technology

[0002] Pertussis (whooping cough) is a highly contagious respiratory disease, primarily caused by Bordetella pertussis. Before the invention of vaccines, it was a leading cause of death in children. Vaccination is an effective means of preventing and controlling pertussis. Pertussis vaccines can be divided into whole-cell pertussis vaccines (wP) and acellular pertussis vaccines (aP). wP involves inactivating whole bacteria through high temperature, Malformin, or formaldehyde; however, this technology has been phased out in my country due to the serious adverse reactions it can cause. aP, based on purification processes, can be divided into co-purified vaccines and component-purified vaccines. Co-purified vaccines are purified by sucrose density gradient centrifugation to obtain an antigen mixture solution (containing pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN), etc.), which can only roughly control the total amount and antigen ratio. Component-purified vaccines are purified by column chromatography to extract antigen components, which are then mixed in a certain proportion to precisely control the content of effective antigen components.

[0003] PT is the most important antigen for vaccine efficacy. Whether used alone or in combination with other antigens, it effectively prevents pertussis and is an essential component of the pertussis vaccine. PT has a molecular weight of approximately 105 kDa and is an AB-type toxin composed of five subunits: S1, S2, S3, S4, and S5. The A-type toxin is composed of a single S1 subunit, while the B-type oligomer is a pentamer composed of S2, S3, S5, and two S4 subunits. PT possesses various biological activities, such as leukocytosis, histamine sensitization, enhanced vasoactive substance allergy, and pancreatic islet activation. Most of these biological activities are related to the ADP-ribosyltransferase activity of the S1 subunit.

[0004] Because PT possesses various biological activities, it must be detoxified before it can be used in vaccine preparation. Chinese patent CN117844719A discloses an attenuated recombinant strain of Bordetella pertussis, its construction method, and its application. However, the recombinant strains constructed contain the kan resistance gene. The PT protein content secreted by the recombinant strains FE3 and FE16 increased with increasing culture time during continuous culture, with maximum values ​​of approximately 800 ng / mL and 200 ng / mL, respectively; the detection value of recombinant strain 18123 remained consistently below the detection limit. Furthermore, in large-scale vaccine production, strains carrying resistance genes may lead to widespread drug resistance problems. If bacteria in the human body acquire resistance genes, commonly used antibiotics may become ineffective, increasing treatment difficulty and drug safety risks. Secondly, recombinant bacteria containing resistance genes may affect PT protein expression. This patent did not conduct experiments related to protein integrity, and it cannot be determined whether recombinant bacteria containing resistance genes can secrete complete PT proteins containing S1, S2, S3, S4, and S5 subunits. Summary of the Invention

[0005] In view of this, the present invention provides a recombinant CS bacterium of attenuated pertussis, its construction method and application. The recombinant CS bacterium constructed by reverse screening on DOG plates does not contain the resistance gene, thus ensuring the efficient expression of PT protein.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A recombinant CS strain of attenuated pertussis, wherein the recombinant CS strain is obtained by mutating the 9th arginine residue of the S1 subunit to a lysine residue and the 129th glutamic acid residue to a glycine residue in the CS strain with accession number CMCC 58003. The amino acid sequence of the S1 subunit of the recombinant CS strain is shown in SEQ ID NO.1. SEQ ID NO.1: MRCTRAIRQTARTGWLTWLAILAVTAPVTSPAWADDPPATVYKYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFY GAASSYFEYVDTYGDNAGRILAGALATYQSGYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRMAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSF.

[0007] Furthermore, the genome of the CS recombinant bacteria contains a nucleotide sequence as shown in SEQ ID NO.2 or a sequence encoding an amino acid sequence as shown in SEQ ID NO.1 and having genetic code degeneracy; SEQ ID NO.2:

[0008] A method for constructing the above-mentioned CS recombinant bacteria includes the following steps: S1. Prepare recombinant plasmid gPT-pCC1 containing the mutant gene of the S1 subunit of PT protein, the Amp selection gene, and the Galk selection gene; S2. Prepare competent cells of wild-type pertussis CS strain and electroporate plasmid gPT-pCC1 into competent cells of wild-type pertussis CS strain; S3. Prepare BG sheep blood screening plates containing Amp, inoculate competent cells from S2 and screen out a round of recombinant pertussis CS strains. S4. Prepare BG sheep blood screening plates containing DOG, inoculate with the positive strains selected in S3 and screen again to obtain the CS recombinant bacteria. S5. Prepare competent cells from the recombinant pertussis CS bacteria in S4, and electroporate the plasmid gPT-pCC1 into the competent cells of the recombinant pertussis CS bacteria. Then, perform the same screening steps as in steps S3 and S4 to obtain the recombinant CS bacteria.

[0009] In some specific embodiments, preferably, the nucleotide sequence of the target fragment in the recombinant plasmid gPT-pCC1 in step S1 is as shown in SEQ ID NO.3.

[0010]

[0011] Furthermore, the preparation of competent cells of wild-type pertussis CS strain in step S2 is as follows: S21. The wild-type pertussis CS strain was cultured to the logarithmic growth phase, centrifuged, and the bacterial cells were collected. Then, the cells were resuspended and washed with sterile double-distilled water at 2-8℃, centrifuged, and the bacterial cells were collected. S22. Resuspend the bacterial cells obtained in the previous step in a glycerol solution at 2~8℃, let stand on ice, centrifuge, and collect the bacterial cells; S23. Resuspend the bacterial cells obtained in the previous step in a glycerol solution at 2~8℃, dispense 100μL per vial on ice, and store at -80℃ for later use.

[0012] In some specific embodiments, preferably, the centrifugation conditions in steps S21, S22, and S23 are the same: centrifugation at 4000 rpm for 15 min at 4°C. In steps S22 and S23, the volume concentration of the glycerol solution is 10%. The operation in step S22 is repeated twice.

[0013] In some specific embodiments, preferably, the electro-spinning parameters in step S2 are set as follows: voltage 2.5kV, resistance 200Ω, capacitance 25µF, electro-spinning time not exceeding 5ms, and electrode spacing of the electro-spinning cup 2mm.

[0014] Furthermore, in step S3, the concentration of Amp is 100 μg / mL, and the culture time is 2-3 days.

[0015] Furthermore, in step S4, the DOG concentration is 2% and the culture time is 4-5 days.

[0016] The above-mentioned CS recombinant bacteria are used in the preparation of products for the treatment and / or detection and / or prevention of pertussis.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention successfully constructed a deletion screening nucleotide sequence. ampr Genes and galk A double-mutated *Pertussis pertussis* CS strain was constructed (the 9th arginine residue of the PT protein S1 subunit was mutated to a lysine residue (R9K, CGC→AAG), and the 129th glutamic acid residue was mutated to a glycine residue (E129G, GAA→GGG)). The constructed recombinant strain exhibited a growth rate similar to that of the wild-type *Pertussis pertussis* CS strain. Both the secreted PT protein and gPT protein clearly showed five distinct subunits. The maximum gPT protein content was 1049 ng / mL. The toxicity was reduced by 3.125 × 10⁻⁶ compared to the PT protein obtained from the purified wild-type *Pertussis pertussis* CS strain. 4 times.

[0018] The R9K / E129G double mutant pertussis CS strain prepared in this study does not carry the resistance gene and will not lead to drug resistance issues. The integrity of the PT protein was verified by SDS-PAGE, which showed that it contains S1, S2, S3, S4, and S5 subunits. The in vitro toxicity of the PT protein was detected by CHO cell agglutination assay, which confirmed that the toxicity of the PT protein was significantly reduced. The strain used was the Bordetella pertussis CS strain used in the production of pertussis vaccines in China, which lays the foundation for the development of novel pertussis vaccines. Attached Figure Description

[0019] Figure 1 This is the plasmid map of gPT-pCC1 in Example 1 of the present invention.

[0020] Figure 2 This is a PCR verification result of the first round of recombination of Pertussis CS in Example 1 of the present invention (where M: 5000bp DNA Maker; lanes 1 and 3: wild-type Pertussis CS; lane 2: upstream fragment obtained by PCR of the first recombinant bacterial culture grown on BG solid plates containing 100µg / mL Amp using CZ-F / CZ-R primers; lane 4: downstream fragment obtained by PCR of the first recombinant bacterial culture grown on BG solid plates containing 100µg / mL Amp using XY-F / XY-R primers).

[0021] Figure 3 This is a PCR verification result of the second round of recombination of Pertussis CS in Example 1 of the present invention (where M: 5000bp DNA Maker; lanes 1-6: Pertussis CS bacterial culture after secondary recombination).

[0022] Figure 4 This is a PCR verification result of the third round of recombination of *C. pertussis* CS in Example 1 of the present invention (where M: 5000bp DNA Maker; lanes 1 and 3: wild-type *C. pertussis* CS; lane 2 is the upstream fragment obtained by PCR using CZ-F / CZ-R primers for the three-round recombination bacterial culture grown on BG solid plates containing 100 µg / mL Amp; lane 4 is the downstream fragment obtained by PCR using XY-F / XY-R primers for the three-round recombination bacterial culture grown on BG solid plates containing 100 µg / mL Amp).

[0023] Figure 5 This is a PCR verification result of the fourth round of recombination of Pertussis CS in Example 1 of the present invention (where M: 5000bp DNA Maker; lanes 1-6: Pertussis CS bacterial culture after four rounds of recombination).

[0024] Figure 6The growth curves of the R9K / E129G double mutant pertussis CS strain and the wild-type pertussis CS strain in Example 2 of the present invention are shown.

[0025] Figure 7 The PT protein expression curves of the R9K / E129G double mutant pertussis CS strain and the wild-type pertussis CS strain in Example 2 of this invention are shown.

[0026] Figure 8 This is an SDS-PAGE comparison of the PT protein purified from the wild-type pertussis CS strain in Example 2 of the present invention and the gPT protein purified from the gene-edited R9K / E129G double mutant pertussis CS strain (where M: 180 kDa protein maker; lane 1: wild-type pertussis CS strain PT protein; lane 2: gene-edited R9K / E129G double mutant pertussis CS strain gPT protein).

[0027] Figure 9 This is a schematic diagram of CHO cell clusters in Embodiment 2 of the present invention.

[0028] Figure 10 This is a graph showing the results of the CHO cell agglutination assay for detecting the in vitro toxicity of PT protein in Example 2 of this invention.

[0029] Figure 11 This is a schematic diagram of homologous recombination gene editing in the technical solution of the present invention. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0031] Example 1 This embodiment provides an attenuated recombinant pertussis CS strain constructed based on the CS strain with accession number CMCC 58003. The specific construction is as follows (see schematic diagram of homologous recombination gene editing). Figure 11 ): 1.1 Construction of plasmid gPT-pCC1 Using pCC1 as a vector, and BamHI and PmeI restriction enzyme sites as ligation sites for the target fragment, the target fragment contains the PT protein-S1 subunit double mutant gene, the ampr gene, and the galk gene. The target fragment is integrated into the pCC1 plasmid through gene synthesis to obtain the gPT-pCC1 plasmid. The plasmid map is shown below. Figure 1 The nucleotide sequence of the target fragment is shown in SEQ ID NO.3.

[0032] 1.2 Preparation of competent cells of wild-type cough CS strain 100 mL of *Pertussis CS* strain was cultured to the logarithmic growth phase. The bacterial suspension was collected and aliquoted into two 50 mL sterile centrifuge tubes. The tubes were centrifuged at 4000 rpm for 15 min at 4°C, and the waste liquid was discarded, retaining the bacterial cells. Each tube was resuspended in 20 mL of pre-chilled sterile double-distilled water (2–8°C) and washed. The tubes were then centrifuged at 4000 rpm for 15 min at 4°C, and the waste liquid was discarded, retaining the bacterial cells. Each tube was then resuspended in 10 mL of pre-chilled 10% (v / v) glycerol (2–8°C), incubated on ice for 10 min, and then centrifuged at 4°C for 4000 rpm. Centrifuge at rpm for 15 min, discard the waste liquid and retain the bacterial cells; resuspend the bacterial cells in 5 mL of 10% (V / V) glycerol pre-cooled at 2-8℃ in each tube, let stand on ice for 10 min, then centrifuge at 4℃ and 4000 rpm for 15 min, discard the waste liquid and retain the bacterial cells; resuspend the bacterial cells in 1 mL of 10% (V / V) glycerol pre-cooled at 2-8℃ in each tube, aliquot 100 μL into 1.5 mL sterile centrifuge tubes on ice, and store at -80℃ for later use.

[0033] 1.3 Electroporation transformation of pertussis CS strains Thaw 100 μL of competent cells on ice, add 1 µg of plasmid gPT-pCC1, and gently mix by pipetting. Pre-cool the electroporation cuvette on ice, add the electroporation system to the cuvette, wipe the surface of the cuvette dry, and electroporate. The electroporation parameters are set as follows: voltage 2.5 kV, resistance 200 Ω, capacitance 25 µF, electroporation time not exceeding 5 ms, and electrode spacing of the electroporation cuvette 2 mm. Immediately after electroporation, add 1 mL of preheated 37°C SSM liquid medium for recovery. Transfer the system to a 50 mL sterile centrifuge tube, add another 2 mL of preheated 37°C SSM liquid medium, and incubate overnight at 37°C and 220 rpm on a shaker for recovery. After recovery, centrifuge at 4000 rpm for 10 min, discard the supernatant, add 100 µL of SSM liquid medium, resuspend the cells, and spread them on BG solid plates containing 100 µg / mL Amp. Incubate the plates at 37°C and observe the growth of clones.

[0034] 1.4 One round of recombination of C. pertussis (1) Use a sterile pipette tip to pick up the colonies that grow on the plate and inoculate them into SSM liquid medium containing Amp and culture for 2-3 days. Take the bacterial solution for PCR verification.

[0035] (2) The PCR primers are shown below: Table 1 Primer sequences for the first round of recombinant PCR validation

[0036] (3) The bacterial culture PCR reaction system is shown below: Table 2. PCR reaction system for a single recombinant bacterial culture

[0037] (4) The bacterial culture PCR reaction procedure is as follows: Table 3. Procedure for a single recombinant bacterial culture PCR reaction

[0038] (5) Prepare a 1% agarose gel, and perform gel electrophoresis on the obtained PCR products. Observe the electrophoresis results using a gel imaging system.

[0039] (6) Results: The gel electrophoresis results are as follows Figure 2 As shown, lanes 1 and 3 contain wild-type *C. pertussis* CS bacteria; lanes 2 and 4 contain recombinant bacterial cultures grown on BG solid plates containing 100 µg / mL Amp. Lane 2 contains the upstream fragment obtained by PCR using SY-F / SY-R primers, with a theoretical PCR fragment length of 3402 bp; lane 4 contains the downstream fragment obtained by PCR using XY-F / XY-R primers, with a theoretical PCR fragment length of 3696 bp. Bands appear in lanes 2 and 4 at approximately 3000 bp, consistent with the theoretical length of the bacterial culture PCR fragment, indicating that the plasmid was successfully electroporated and underwent the first round of recombination. Forward selection was completed on BG solid plates containing 100 µg / mL Amp, and the target nucleotide sequence PT protein-S1 subunit double mutant gene, the selected nucleotide sequences ampr gene, and galk gene were integrated into wild-type *C. pertussis* CS bacteria through homologous recombination.

[0040] (7) The upstream and downstream PCR fragments were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed that the glutamic acid residue at position 129 of the PT protein-S1 subunit in the upstream PCR fragment was mutated to a glycine residue (E129G, Glu→Gly), and the arginine residue at position 9 of the PT protein-S1 subunit in the downstream PCR fragment was mutated to a lysine residue (R9K, Arg→Lys).

[0041] 1.5 Pertussis CS bacteria undergoes a second round of recombination (1) The recombinant pertussis CS strain from 1.4 was cultured in SSM liquid medium for 2-3 days. The bacterial solution was then dipped into a spreader and streaked onto a BG plate containing 2% DOG for 5-7 days. Colonies grown on the BG plate were picked up with a sterile pipette tip and inoculated into SSM liquid medium for 2-3 days. The bacterial solution was then aspirated for bacterial PCR.

[0042] (2) The PCR primers are shown below: Table 4 Primer sequences for the second round of recombinant PCR

[0043] (3) The bacterial culture PCR reaction system is shown below: Table 5. PCR reaction system for secondary recombinant bacterial culture

[0044] (4) The bacterial culture PCR reaction procedure is as follows: Table 6. PCR reaction procedure for secondary recombinant bacterial culture

[0045] (5) Prepare a 1% agarose gel, and perform gel electrophoresis on the obtained PCR products. Observe the electrophoresis results using a gel imaging system.

[0046] (6) Results: The gel electrophoresis results are as follows Figure 3 As shown, lanes 1-6 all contain *C. pertussis* CS strains grown on BG solid plates containing 2% DOG. The theoretical length of the bacterial PCR fragment is 3282 bp. Bands appear in lanes 1-6 at approximately 3000 bp, consistent with the theoretical length of the bacterial PCR fragment, indicating that the second round of recombination was successfully completed on BG solid plates containing 2% DOG. The reverse selection was completed on BG solid plates containing 2% DOG. Strains 1-6 lack the selection nucleotide sequences ampr and galk genes.

[0047] (7) The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed that the 9th arginine residue of the S1 subunit of the PT protein of strain 1 was mutated to a lysine residue (R9K, CGC→AAG), indicating that the strain was an R9K single mutant pertussis strain; the 129th glutamic acid residue of the S1 subunit of the PT protein of strains 2, 5, and 6 was mutated to a glycine residue (E129G, GAA→GGG), indicating that the strain was an E129G single mutant pertussis strain; strains 3 and 4 did not mutate.

[0048] 1.6 Three-times recombinant pertussis CS strains were screened out Following the steps in 1.2-1.5 above, the R9K single mutant recombinant pertussis strains selected in 1.5 were screened to obtain the tertiary recombinant pertussis CS strains.

[0049] The specific procedures for the third round of recombination of C. pertussis bacteria are as follows: (1) Use a sterile pipette tip to pick up the colonies that have grown on the petri dish and inoculate them into SSM liquid medium containing 100 μg / mL Amp for 2-3 days. Take the bacterial solution for PCR verification.

[0050] (2) The PCR primers are shown below: Table 7 Primer sequences for the third round of recombinant PCR validation

[0051] (3) The bacterial culture PCR reaction system is shown below: Table 8. Three-stage recombinant bacterial culture PCR reaction system

[0052] (4) The bacterial culture PCR reaction procedure is as follows: Table 9. Procedure for three recombinant bacterial culture PCR reactions

[0053] (5) Prepare a 1% agarose gel, and perform gel electrophoresis on the obtained PCR products. Observe the electrophoresis results using a gel imaging system.

[0054] (6) Results: The gel electrophoresis results are as follows Figure 4 As shown, the gel electrophoresis results are as follows: Figure 2 As shown, lanes 1 and 3 contain wild-type *C. pertussis* CS bacteria; lanes 2 and 4 contain three-stage recombinant bacterial cultures grown on BG solid plates containing 100 µg / mL Amp. Lane 2 contains the upstream fragment obtained by PCR using SY-F / SY-R primers, with a theoretical PCR fragment length of 3402 bp; lane 4 contains the downstream fragment obtained by PCR using XY-F / XY-R primers, with a theoretical PCR fragment length of 3696 bp. Bands appear at approximately 3000 bp in lanes 2 and 4, consistent with the theoretical length of the bacterial culture PCR fragment, indicating that the plasmid was successfully electroporated and underwent a third round of recombination. Forward selection was completed on BG solid plates containing 100 µg / mL Amp, and the target nucleotide sequence PT protein-S1 subunit double mutant gene, the selected nucleotide sequences ampr gene, and galk gene were integrated into wild-type *C. pertussis* CS bacteria through homologous recombination.

[0055] 1.7 Four recombinant pertussis CS strains (i.e., target recombinant bacteria) were screened out. Following steps 1.3-1.5 above, the three recombinant pertussis CS strains screened in 1.6 were processed.

[0056] The specific procedures for the fourth round of recombination of C. pertussis are as follows: (1) The pertussis CS strain recombined three times in 1.6 was cultured in SSM liquid medium for 2-3 days. The bacterial solution was dipped into a spreader and streaked onto BG plates containing 2% DOG for 5-7 days. Colonies grown on BG plates were picked up with a sterile pipette tip and inoculated into SSM liquid medium for 2-3 days. The bacterial solution was then aspirated for bacterial PCR.

[0057] (2) The PCR primers are shown below: Table 10 Primer sequences for the fourth round of recombinant PCR

[0058] (3) The bacterial culture PCR reaction system is shown below: Table 11 Four Recombinant Bacterial Culture PCR Reaction Systems

[0059] (4) The bacterial culture PCR reaction procedure is as follows: Table 12 Procedure for Four Recombinant Bacterial Culture PCR Reactions

[0060] (5) Prepare a 1% agarose gel, and perform gel electrophoresis on the obtained PCR products. Observe the electrophoresis results using a gel imaging system.

[0061] (6) Results: The gel electrophoresis results are as follows Figure 5 As shown, lanes 1-6 all contain *Pertussis pertussis* CS strains grown on BG solid plates containing 2% DOG. The theoretical length of the bacterial culture PCR fragment is 3282 bp. Bands appear in lanes 1-6 at approximately 3000 bp, consistent with the theoretical length of the bacterial culture PCR fragment, indicating that the fourth round of recombination was successfully completed on BG solid plates containing 2% DOG. The reverse selection was completed on BG solid plates containing 2% DOG. Strains 1-6 lack the selection nucleotide sequences ampr gene and galk gene.

[0062] (7) The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed that the 9th arginine residue of the S1 subunit of the PT protein of strains 1, 3, and 6 was mutated to a lysine residue (R9K, CGC→AAG), indicating that the R9K single mutant pertussis CS strain was successfully obtained; the 9th arginine residue of the S1 subunit of the PT protein of strains 2, 4, and 5 was mutated to a lysine residue (R9K, CGC→AAG), and the 129th glutamic acid residue was mutated to a glycine residue (E129G, GAA→GGG), indicating that the R9K / E129G double mutant pertussis CS strain was successfully obtained.

[0063] 1.8 Extraction and validation of the genome of the R9K / E129G double mutant recombinant bacteria. Transfer the overnight cultured R9K / E129G double mutant *C. cephalosporinus* to a centrifuge tube, centrifuge at 10,000 rpm for 3 seconds, and discard the supernatant. Add 180 µL of Buffer ACL and 20 µL of Proteinase K to the bacterial pellet, vortex thoroughly, and incubate at 56 °C for 1 h. Add 200 µL of Buffer CL and vortex thoroughly. Add 200 µL of anhydrous ethanol and mix thoroughly again. Transfer the mixture to an EZ-10 Spin Column placed in a 2 mL collection tube. Centrifuge at 12,000 rpm for 1 min. Discard the liquid in the collection tube. Add 500 µL of CW1 Solution and centrifuge at 12,000 rpm for 1 min. Discard the liquid in the collection tube. Add 500 µL of CW2 Solution and centrifuge at 12,000 rpm for 1 min. Discard the liquid in the collection tube. Place the empty adsorption column back into the centrifuge tube and centrifuge at 12,000 rpm for 2 min to dry the adsorption membrane. Place the adsorption column into a clean 1.5 mL centrifuge tube. Open the column and let it stand at room temperature for 2-3 minutes until the ethanol has completely evaporated. Add 50-100 µL of Buffer CE directly to the center of the adsorption membrane. Let it stand at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes to elute the DNA. Send the DNA to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were consistent with those of Example 9. The 9th arginine residue of the S1 subunit of the PT protein was mutated to a lysine residue (R9K, CGC→AAG), and the 129th glutamic acid residue was mutated to a glycine residue (E129G, GAA→GGG). The ampr gene and galk gene, which are the screening nucleotide sequences, were also missing, indicating that the R9K / E129G double mutant Japanese cough CS strain was successfully obtained.

[0064] Example 2 This embodiment investigated the PT protein expression and toxicity of the recombinant CS bacteria obtained in Example 1 as follows: 2.1 Growth curve of R9K / E129G double mutant recombinant bacteria Wild-type *C. pertussis* and R9K / E129G double mutant *C. pertussis* strains were inoculated into SSM liquid medium and cultured until OD. 600nm =1.0, 1 mL of wild-type *C. pertussis* CS strain and R9K / E129G double mutant *C. pertussis* CS strain were respectively inoculated into 500 mL shake flasks containing 100 mL of SSM liquid medium, placed in a bacterial shaker, and cultured at 37℃ and 200 rpm. Bacterial suspensions were collected at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 60 h, and 72 h, and the OD of the bacterial suspensions was measured. 600nm The numerical values ​​were plotted as curves, and the results are as follows: Figure 6As shown, the growth rate of the R9K / E129G double mutant recombinant bacteria is similar to that of the wild-type pertussis CS strain.

[0065] 2.2 Expression curve of PT protein after gene detoxification Wild-type *C. pertussis* and R9K / E129G double mutant *C. pertussis* strains were inoculated into SSM liquid medium and cultured until OD. 600nm =1.0, 1 mL of wild-type pertussis CS and R9K / E129G double mutant pertussis CS were inoculated into 500 mL shake flasks containing 100 mL of SSM liquid medium, placed in a bacterial shaker, and incubated at 37 ℃ and 200 rpm. Bacterial culture was collected at 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, and 48 h, respectively. The culture was centrifuged at 12000 rpm for 8 minutes, and the supernatant was collected. The supernatant was detected using a double-antibody sandwich ELISA method. The supernatant was diluted 10, 20, 40, 80, 160, 320, 640, and 1280 times. The WHO first-generation pertussis toxin standard JNIH-5 was diluted to concentrations of 20, 10, 5, 2.5, and 1.25 ng / mL. The corresponding values ​​of the samples within this range were multiplied by the dilution factor, and the arithmetic mean was calculated to obtain the PT content in the collected culture supernatant. The time-effect curve was plotted accordingly. Figure 7 As shown, the maximum gPT protein content secreted by the R9K / E129G double mutant pertussis CS strain was 1049 ng / mL, while the maximum PT protein content secreted by the wild-type pertussis CS strain was 2023 ng / mL. The PT protein content secreted by the R9K / E129G double mutant pertussis CS strain was much lower than that of the wild-type pertussis CS strain.

[0066] 2.3 SDS-PAGE analysis of PT protein integrity The PT protein was obtained by purifying wild-type *C. pertussis* CS strain, and the gPT protein was obtained by gene-editing the R9K / E129G double mutant *C. pertussis* CS strain. 10 μg of PT protein and gPT protein were added to a reducing 5×SDS loading buffer and boiled at 100℃ for 10 min. The electrophoresis gel was fixed on the electrophoresis apparatus, and electrophoresis buffer was added. Protein markers and samples were added to the electrophoresis gel. The power was turned on, and the gel was run at a constant current of 30 mA until the bromophenol blue indicator was near the bottom of the precast gel. The electrophoresis gel was then removed, stained with Coomassie Brilliant Blue staining solution for 2 h, and then destained in destaining solution before photographing.

[0067] The results are as follows Figure 8As shown, both the PT protein and the gPT protein have five clearly distinguishable subunits. The theoretical molecular weights of the five subunits S1, S2, S3, S4 and S5 are 26 kDa, 22 kDa, 21 kDa, 12 kDa and 11 kDa, respectively. The size of the SDS-PGAE is consistent with the theoretical value, indicating that the gPT protein was successfully purified and the protein is intact.

[0068] 2.4 CHO cell agglutination assay to detect PT protein toxicity in vitro After culturing CHO cells for 48 hours, trypsin was added for digestion, followed by the addition of DMEM / F12 cell culture medium. The cells were then pipetted until they were in single suspension, and the concentration was diluted to 5 × 10⁻⁶. 4 Add 100 μL / well to a 96-well cell plate and incubate at 37°C for 16–24 h. Dilute PT standard to 0.32 IU / mL as the standard. Use DMEM / F12 cell culture medium as the negative control. Dilute PT protein purified from wild-type pertussis CS strain to 16 ng / mL as the positive control. Dilute gPT protein purified from R9K / E129G double mutant pertussis CS strain to 500 ug / mL as the test sample. Add 250µL of each sample to the first well of a blank 96-well cell culture plate. Add 125µL of DMEM / F12 cell culture medium to each of the remaining wells. Using a pipette, transfer 125µL of sample from each well in the first column to the second column and mix thoroughly. Transfer 125µL of the liquid to the third column and mix thoroughly. Serially dilute up to the twelfth column. Transfer 100µL of the diluted liquid to a 96-well cell culture plate containing pre-incubated and adherent CHO cells. After incubation at 37°C for 48 h, observe the cell clustering of the 96-well cell plate under a microscope. If no cells cluster, the plate is considered "non-clustered". Clusters of less than 50% of cells are considered suspicious ("±"); clusters of 50% or more of cells are considered clusters ("+"); and clusters of 100% of cells are considered obvious clusters ("++"). See the diagram for a schematic of cell clusters. Figure 9 Effective agglutination was defined as CHO cell aggregation of 50% or higher, with a maximum dilution factor for CHO cell aggregation below 50% as the endpoint. Sample toxicity results were calculated using the concentration obtained from the final clustering well dilution factor of the PT standard in each 96-well plate as a control. The calculation formula was: Sample activity (IU / mg) = PT standard activity (IU / mL) / Sample clustering endpoint concentration (mg / mL).

[0069] The results are as follows Figure 10 As shown: the in vitro toxicity result of PT protein purified from wild-type pertussis CS strain was 10000 IU / mg, while the in vitro toxicity result of PT protein purified from four recombinant pertussis CS strains was 0.32 IU / mg, representing a decrease in toxicity of 3.125 × 10⁻⁶ IU / mg. 4 times.

[0070] The above series of studies demonstrate that this application has successfully obtained a pertussis CS strain with R9K / E129G double mutation, which can be used for the research and development and production of aP component purified vaccines.

[0071] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0072] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant attenuated pertussis CS bacterium, characterized in that, The CS recombinant bacteria were obtained by mutating the 9th arginine residue of the S1 subunit to a lysine residue and the 129th glutamic acid residue to a glycine residue in the CS strain with accession number CMCC58003. The amino acid sequence of the S1 subunit of the CS recombinant bacteria is shown in SEQ ID NO.

1.

2. The CS recombinant bacteria according to claim 1, characterized in that, The genome of the CS recombinant bacteria contains a nucleotide sequence as shown in SEQ ID NO.2 or a sequence that encodes an amino acid sequence as shown in SEQ ID NO.1 and has genetic code degeneracy.

3. A method for constructing the CS recombinant bacteria according to claim 1 or 2, characterized in that, Includes the following steps: S1. Prepare recombinant plasmid gPT-pCC1 containing the mutant gene of the S1 subunit of PT protein, the Amp selection gene, and the Galk selection gene; S2. Prepare competent cells of wild-type pertussis CS strain and electroporate plasmid gPT-pCC1 into competent cells of wild-type pertussis CS strain; S3. Prepare BG sheep blood screening plates containing Amp, inoculate competent cells from S2 and screen to obtain a recombinant pertussis CS strain. S4. Prepare BG sheep blood screening plates containing DOG, inoculate with the positive strains selected in S3 and screen again to obtain secondary recombinant pertussis CS bacteria. S5. Prepare competent cells from the recombinant pertussis CS bacteria in S4, and electroporate the plasmid gPT-pCC1 into the competent cells of the recombinant pertussis CS bacteria. Then, perform the same screening steps as in steps S3 and S4 to obtain the recombinant CS bacteria.

4. The construction method according to claim 3, characterized in that, The nucleotide sequence of the target fragment in the recombinant plasmid gPT-pCC1 in step S1 is shown in SEQ ID NO.

3.

5. The construction method according to claim 3, characterized in that, The preparation of competent cells of wild-type pertussis CS strain in step S2 is as follows: S21. The wild-type pertussis CS strain was cultured to the logarithmic growth phase, centrifuged, and the bacterial cells were collected. Then, the cells were resuspended and washed with sterile double-distilled water at 2-8℃, centrifuged, and the bacterial cells were collected. S22. Resuspend the bacterial cells obtained in the previous step in a glycerol solution at 2~8℃, let stand on ice, centrifuge, and collect the bacterial cells; S23. Resuspend the bacterial cells obtained in the previous step in a glycerol solution at 2~8℃, dispense 100μL per vial on ice, and store at -80℃ for later use.

6. The construction method according to claim 5, characterized in that, The centrifugation conditions in steps S21, S22, and S23 are all the same: centrifuge at 4000 rpm for 15 min at 4℃; In steps S22 and S23, the volume concentration of the glycerol solution is 10%. The operation in step S22 is repeated twice.

7. The construction method according to claim 3, characterized in that, The electro-spinning parameters in step S2 are set as follows: voltage 2.5kV, resistance 200Ω, capacitance 25µF, electro-spinning time not exceeding 5ms, and electrode spacing of the electro-spinning cup 2mm.

8. The construction method according to claim 3, characterized in that, In step S3, the concentration of Amp is 100 μg / mL, and the culture time is 2-3 days.

9. The construction method according to claim 3, characterized in that, In step S4, the DOG concentration is 2% and the incubation time is 4-5 days.

10. The use of the CS recombinant bacteria of claim 1 or 2 in the preparation of products for the treatment and / or detection and / or prevention of pertussis.

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