Recombinant expression vector for pertussis antigens, genetically engineered strains thereof and their uses
Patent Information
- Application Number
- JP2024536245
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2021-12-17
- Filing Date
- 2022-11-10
- Publication Date
- 2025-10-22
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Abstract
Description
[Technical field]
[0001] The present invention belongs to the field of biopharmaceuticals, and relates to a recombinant expression vector for pertussis antigen, its genetically engineered strain and its use. Specifically, the present invention relates to a recombinant expression vector for overexpressing pertactin antigen and simultaneously expressing pertussis toxin antigen, a genetically engineered strain containing the same, and the use of both in producing pertactin and pertussis toxin antigens. [Background technology]
[0002] Whooping cough is a severe respiratory infection caused by Bordetella pertussis, which causes great harm to infants and young children. Its clinical characteristics are a cough that gradually becomes more severe, typically paroxysmal and spasmodic, and a long, deep inspiratory whoosh that sounds like a rooster crowing at the end of the cough. It is called whooping cough because it lasts for 2 to 3 months.
[0003] Currently, the main method of preventing whooping cough is vaccination. The first generation whole cell pertussis vaccine (wPV) has been able to control the disease well, but it is being replaced by the safer acellular pertussis vaccine (aPV) because of serious side effects caused by vaccination. However, since 2010, the incidence of whooping cough in adults and adolescents has increased again, and analysis has found that the main reasons for this include the rapid decline in the effectiveness of aPV and the mutation of strains due to selective pressure caused by the vaccine. Therefore, the continuous improvement of the whooping cough vaccine is an effective measure to combat the spread of whooping cough infection.
[0004] During the growth process of Bordetella pertussis bacteria, various antigens are produced, including Pertussis Toxin (PT), Filamentous Haemagglutinin (FHA), Pertactin (PRN), and Lipopolysaccharide (LPS). All domestically produced aPV sold in Korea is made by purifying pertussis antigen solution using the co-purification method, treating it with an antidote, and then adsorbing it to an aluminum salt adjuvant. As the different antigen components of pertussis are not separated separately, there is a large difference in the ratio of various antigens in the pertussis antigen solution depending on the batch, which affects the stability of the vaccine quality.
[0005] The imported aPV uses more advanced column chromatography to extract and detoxify antigens such as PT and FHA separately, and then mix these antigen components in a specified ratio to produce the vaccine. This process ensures the consistency of the quality of each batch of vaccine, and the antigen purity is 90%-95%, making it suitable for the development of DTacP-based mixed vaccines (Hu Yiqin et al., Establishment of a Novel Process for Purifying Acellular Component Whooping Cough Vaccine). For example, Chinese patent CN102793915B provides a three-component pertussis vaccine composition, namely, PT, PRN and FHA are purified respectively and mixed in a specified ratio. According to related materials, the FHA antigen is easily decomposed, which makes it difficult to evaluate the purification effect. Several publications (Wu Tengjie, Zhang Bin, Zhang Qing et al., Establishment of a novel process for the purification of acellular pertussis vaccine [J], China Journal of Biological Products, 26(01):5-8, etc.) have reported purification methods to separate FHA protein and PT protein, but the step of extracting PT protein alone is complicated.
[0006] The above methods all have the problem of low expression of antigenic proteins and complicated separation and purification. Therefore, it is urgent to develop a method for producing pertussis antigens that can efficiently express antigenic components and has a simplified separation and purification process. Summary of the Invention [Problem to be solved by the invention]
[0007] The present inventors have constructed a pertussis antigen recombinant expression vector and improved a pertussis antigen producing strain to provide a recombinant genetically engineered strain that blocks the expression of FHA protein, so that the PRN protein and PT protein can be easily obtained as a single component. The strain can simultaneously express the PRN protein and the PT protein, and the fermentation time is short, so that the contents of the two proteins produced are significantly higher than those of the wild strain, which is advantageous for separation and purification. [Means for solving the problem]
[0008] In one aspect, the present invention provides a pertussis antigen recombinant expression vector, comprising a pertactin protein expression cassette, a resistance screening gene, and an upstream recombinant nucleic acid fragment and a downstream recombinant nucleic acid fragment of a filamentous hemagglutinin gene, wherein the pertactin protein expression cassette is located between the upstream recombinant nucleic acid fragment and the downstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene, and the upstream recombinant nucleic acid fragment and the downstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene can undergo homologous recombination with the upstream and downstream of the filamentous hemagglutinin gene, respectively, and the filamentous hemagglutinin gene is set forth in SEQ ID NO:1.
[0009] The expression vector of the present invention may be the plasmid pUC57, the pEASY-Blunt cloning vector or a pBBR series vector, and is preferably the plasmid pUC57.
[0010] The recombinant nucleic acid fragment upstream of the filamentous hemagglutinin gene according to the present invention may comprise or be a nucleic acid fragment consisting of consecutive bases from positions m to 1000 of the nucleotide sequence shown in SEQ ID NO:2, where m is a natural number not exceeding 57. Preferably, the recombinant nucleic acid fragment upstream of the filamentous hemagglutinin gene is a nucleic acid fragment consisting of consecutive bases from positions 57 to 1000 of the nucleotide sequence shown in SEQ ID NO:2.
[0011] In one embodiment of the present invention, the nucleotide sequence of the pertactin expression cassette is shown in SEQ ID NO:3.
[0012] The recombinant nucleic acid fragment downstream of the filamentous hemagglutinin gene according to the present invention may comprise or be a nucleic acid fragment consisting of consecutive bases at positions 1 to n of the nucleotide sequence shown in SEQ ID NO:4, where n is a natural number between 798 and 1000. Preferably, the recombinant nucleic acid fragment downstream of the filamentous hemagglutinin gene is a nucleic acid fragment consisting of consecutive bases at positions 1 to 798 of the nucleotide sequence shown in SEQ ID NO:4.
[0013] The resistance screening gene of the present invention may be one or more selected from the group consisting of a kanamycin resistance screening gene, a tetracycline resistance screening gene, an ampicillin resistance screening gene and a chloramphenicol resistance screening gene, and is preferably a kanamycin resistance screening gene. Specifically, the nucleotide sequence of the kanamycin resistance screening gene is shown in SEQ ID NO: 5 (cloning vector pSN-caSAT1, Genebank: MT001914.1), the nucleotide sequence of the tetracycline resistance screening gene is shown in SEQ ID NO: 6 (cloning vector pJC24, Genebank: KC442291.1), the nucleotide sequence of the ampicillin resistance screening gene is shown in SEQ ID NO: 7 (cloning vector pcDNA3-WSN-PB2, Genebank: MT966986.1), and the nucleotide sequence of the chloramphenicol resistance screening gene is shown in SEQ ID NO: 8 (cloning vector pMYC, Genebank: MT572316.1).
[0014] In another aspect, the present invention also provides a genetically engineered recombinant expression of a pertussis antigen comprising a recombinant expression vector according to the present invention.
[0015] In another aspect, the present invention also provides a method for producing a genetically engineered pertussis antigen recombinant expression bacterium of the present invention, comprising transforming a wild-type Bordetella pertussis competent cell with a recombinant expression vector of the present invention.
[0016] In another embodiment of the invention, the wild-type Bordetella pertussis is the wild-type Bordetella pertussis strain ATCC-BAA-589.
[0017] In another embodiment of the present invention, the transformation is electrotransformation, specifically, the electrotransformation may comprise linearizing the recombinant expression vector and electrotransforming wild-type Bordetella pertussis competent cells at 2500V for 5ms.
[0018] In another embodiment of the present invention, the method further comprises screening wild-type Bordetella pertussis competent cells after transformation with the resistance screening gene.
[0019] In yet another aspect, the present invention also provides a method for the production of a pertussis antigen comprising fermentation using a genetically engineered bacterium recombinantly expressing a pertussis antigen according to the present invention.
[0020] In another embodiment of the invention, the fermentation comprises the following steps: (1) The genetically engineered bacteria expressing a recombinant pertussis antigen according to the present invention is inoculated onto a plate containing Bordet-Jung medium and cultured for 40 to 72 hours, preferably 48 hours. (2) The bacterial cells obtained in step (1) are inoculated into a shake flask containing Modified Stainer Scholte medium (MSS medium) and cultured for 22.5 to 25 hours. (3) The bacterial solution obtained in step (2) is inoculated into a fermenter containing MSS medium and cultured for 28 to 30 hours.
[0021] Specifically, the pertussis antigen is pertactin (PRN, the amino acid sequence of which is shown in SEQ ID NO: 9) and / or pertussis toxin (PT, the amino acid sequence of which is shown in SEQ ID NO: 10). SEQ ID NO:9: * sequence number 10:
[0022] The present invention uses a recombinant pertussis antigen expression vector constructed to obtain a genetically engineered Bordetella pertussis bacterium, in which the FHA protein gene is knocked out and the PRN protein gene copies are increased, thereby enabling efficient overexpression of the PRN protein antigen and simultaneous expression of the PT protein. In addition, the PRN protein antigen and the PT protein expressed by the genetically engineered Bordetella pertussis bacterium provided by the present invention are present in the bacterial cells and the fermentation broth supernatant, respectively, which is more advantageous for subsequent antigen separation and purification.
[0023] Compared with some previous reports, the genetically engineered Bordetella pertussis strain OEPRN-PT provided by the present invention has a significant advantage in that it can simultaneously obtain higher expression levels of PRN protein and PT protein. For example, Connor's laboratory has produced a strain expressing two copies of the PRN gene driven by the FHA promoter, and the final PRN protein concentration is about 186.7 μg / mL (Loosmore SM, Yacoob RK, Zealey GR, et al., Hybrid genes over-express pertactin from Bordetella pertussis [J]. Vaccine, 1995, 13(6):571), and the expression concentration of PRN produced by the genetically engineered strain OEPRN-PT provided by the present invention is 418-555 μg / mL. Effect of the Invention
[0024] The present invention demonstrates the following beneficial effects in the examples. 1. By knocking out the FHA gene and blocking the expression of FHA, the effect of FHA protein on PT protein during the antigen purification process of Bordetella pertussis strains was eliminated, and the PT protein content was improved and the purified PT protein contained less contaminating proteins compared to the wild-type strain.
[0025] 2. By increasing the copy number of PRN, the production amount of PRN was significantly increased. Compared with the PRN content of the two-copy strain in the previous report, the PRN protein content of the present invention is significantly higher.
[0026] 3. Compared with the wild type, the genetically engineered strain OEPRN-PT completed its fermentation growth in about 28–30 h, shortening the time by more than 25%.
[0027] 4. The PRN protein expressed by the genetically engineered bacterium OEPRN-PT of the present invention is concentrated in the bacterial body, and the PT protein is concentrated in the bacterial supernatant, whereas both of these proteins produced by the wild-type strain are concentrated in the bacterial supernatant, so the proteins produced by the genetically engineered bacterium OEPRN-PT of the present invention are more suitable for subsequent separation and purification.
[0028] As can be seen from the above, the present invention can simultaneously produce high contents of PRN protein and PT protein from the same B. pertussis genetically engineered strain, and the proteins are easier to separate and purify, thereby meeting the needs for industrial production of antigens. [Brief description of the drawings]
[0029] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. [Figure 1] FIG. 1 is a schematic diagram of the plasmid construction of the recombinant vector pUC57-Fhup-Prn-Fhdown-Kan. [Diagram 2] The resistance gene Kan was linked downstream of the vector fragment Fhup-Prn-Fhdown, and then identified by enzymatic digestion. The resistance gene Kan is approximately 963 bp. In the figure, M is a marker, and lanes 1# and 2# are parallel samples containing the resistance gene Kan. [Diagram 3]This shows the identification of upstream and downstream insertions of recombinant expression vectors by verification at the DNA level, in which M is a marker, lanes 1 to 6 are confirmation of the upstream insertion site, and lanes 1' to 6' are confirmation of the downstream insertion site. [Figure 4] Electrophoresis results for identification of FHA gene knockout by verification at the DNA level, in which M is a marker and lanes 1 to 6 are detection of the FHA gene. [Diagram 5] The PRN protein produced by fermentation of the genetically engineered bacterium OEPRN-PT was applied to a QHP column and purified by elution with Buffer B. [Figure 6] 3 shows the results of HPLC detection of PRN protein purified by a QHP column. [Figure 7] The PT protein produced by fermentation of the genetically engineered bacterium OEPRN-PT was purified by loading onto an SP-inspire column and eluting with Buffer D. [Figure 8] The PT protein produced by fermentation of the wild-type strain WT was applied to an SP-inspire column and purified by elution with Buffer D. Preferred Mode for Carrying Out the Invention
[0030] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent from the description.
[0031] The experimental methods described in this invention are conventional methods unless otherwise stated, and the biological materials referred to may be purchased, unless otherwise stated.
[0032] Example 1: Construction of recombinant vector pUC57-Fhup-Prn-Kan-Fhdown Construct the recombinant vector pUC57-Fhup-Prn-Kan-Fhdown according to the schematic diagram of plasmid construction shown in Figure 1.
[0033] 1.1 Construction of vector fragment Fhup-Prn-Kan-Fhdown The fragment sequences and primers required for constructing the vector fragment Fhup-Prn-Kan-Fhdown are shown in Table 1. Two copies of the PRN gene can be obtained by amplifying each fragment individually and then joining the fragments by overlap PCR.
[0034] 1.2 Construction of recombinant vector pUC57-Fhup-Prn-Kan-Fhdown The vector pUC57 was enzymatically digested using the restriction enzymes HindIII and BamHI. The enzyme digestion system was 200 μL (purchased from NEB), 10 μL of 10×2.1 buffer, 15 μg of plasmid, 4 μL each of HindIII and BamHI, and 200 μL of H2O. The enzyme digestion was carried out at 37° C. for 2 hours, and then the band of interest was detected by 1% agarose electrophoresis and collected. The pUC57 after enzyme digestion was connected to the Fhup-Prn-Kan-Fhdown prepared in step 1.1 using recombinant enzymes (purchased from Nanjing Vazyme Biotech), and the constructed recombinant vector pUC57-Fhup-Prn-Kan-Fhdown was transformed into competent cells of E. coli DH5a strain (purchased from TransGen Biotech), and the primers used were pt-Kan-F and pt-Kan-CZR, respectively (see Table 2), and detected by colony PCR. The results (see Figure 2) show that the Kan resistance fragment was correctly connected and was about 963bp, indicating that the recombinant expression vector pUC57-Fhup-Prn-Kan-Fhdown was successfully constructed. Finally, the positive clones identified as correct by colony PCR were transported to Shanghai Sangon Biotech for sequencing, and the clones whose nucleic acid sequence was completely consistent with the target gene sequence were stored at -80°C.
[0035] [Table 1]
[0036] [Table 2]
[0037] Example 2: Creation of genetically engineered Bordetella pertussis strains The wild-type Bordetella pertussis strain (hereinafter referred to as wild-type strain WT) used in this example was the wild-type Bordetella pertussis strain ATCC-BAA-589 purchased from Kitano Biosciences (BNCC).
[0038] 2.1 Cultivation of fungal cells The glycerol stock of the wild-type strain ATCC-BAA-589 to be made competent was activated, and an appropriate amount of the wild-type strain ATCC-BAA-589 liquid was applied to a Bordet-Jung medium plate and cultured in an incubator at 37°C for 48 hours. After that, the plate was removed, and the plaques on the surface were scraped off with a sampling rod. The plate was inoculated into 100 mL of MSS medium and cultured for 24 hours, and the OD 600 Measure the OD value 600 was limited to 1-3.
[0039] 2.2 Preparation of competent cells The bacterial cells prepared in step 2.1 were collected, centrifuged at 4°C for 15 minutes, resuspended in 100 mL of distilled water pre-cooled at 4°C, washed twice, then centrifuged at 4°C for 15 minutes, and collected. The bacterial cells were resuspended in an appropriate volume of 10% sterile glycerol, washed, centrifuged at 4°C for 15 minutes, collected, and resuspended in 1 mL of 10% sterile glycerol to obtain wild-type strain ATCC-BAA-589 competent cells, which were stored in a refrigerator at -70°C.
[0040] 2.3 Preparation of transformation fragments The recombinant expression vector pUC57-Fhup-Prn-Fhdown-Kan prepared in Example 1 was linearized upstream of the Fhup fragment using HindIII restriction enzyme.
[0041] 2.4 Electrotransformation and homologous recombination 2 µg of linearized fragment DNA was introduced into the wild-type strain ATCC-BAA-589 competent cells prepared in step 2.2 under conditions of 2500 V and 5 ms. After 24 hours of liquid culture, the cells were plated on solid medium containing kanamycin (which may be replaced with tetracycline, ampicillin, chloramphenicol, etc. depending on the resistance gene), cultured for 3 to 5 days, and single clones were picked and verified.
[0042] Example 3: Identification of genetically engineered Bordetella pertussis strains 3.1 DNA level verification 3.1.1 Experimental steps The single clones prepared in Example 2 were picked and placed in 1.5 mL EP tubes containing 10 μL of MSS, and 2 μL was drawn out as a template for PCR verification, while the single clones were streaked.
[0043] [Table 3]
[0044] 3.1.2 Experimental results The verification results showed that both the upstream insertion site and the downstream insertion site were correct (Figure 3), and the FHA gene knockout was identified (Figure 4), indicating that the genetically engineered bacterium OEPRN-PT was successfully constructed (the genetically engineered bacterium OEPRN-PT of the present invention has an increased PRN gene through recombination, and the FHA gene is partially knocked out, so that the FHA protein is not expressed).
[0045] 3.2 Growth and fermentation verification 3.2.1 Experimental steps 100 μL of the genetically engineered bacteria OEPRN-PT verified as correct in step 3.1 was inoculated onto two (parallel experiments) plates containing Bordet-Jung medium and incubated at 37 °C for approximately 48 h. Microscopic examination revealed no bacterial contamination. The bacterial lawn was scraped off from the plate and inoculated into 300 mL of MSS medium, and the OD 600The value was measured and found to be 2.12, and no bacterial contamination was found by microscopic examination. 300 mL of the cultured bacterial liquid was taken and inoculated into a fermenter containing 3 L of MSS medium. The initial fermentation conditions were 35°C, 150 rpm, and 2 L / min ventilation, and the dissolved oxygen was limited to 40% during fermentation. After 29 hours of fermentation, the culture was terminated according to the recovery of dissolved oxygen, and the bacterial sediment was collected.
[0046] 3.2.2 Experimental results The genetically engineered bacteria OEPRN-PT and the wild-type strain WT were fermented, and the results were compared and analyzed (see Table 4). The growth time of the wild-type strain WT was about 36 to 42 hours, while the growth time of the genetically engineered bacteria OEPRN-PT in the fermenter stage was about 28 to 30 hours, which was shortened by more than 25%. The concentration of PRN protein produced by the genetically engineered bacteria OEPRN-PT was 418 to 555 μg / mL, and PRN protein was not detected when the wild-type strain was harvested. The concentration of PT protein produced by the genetically engineered bacteria OEPRN-PT was about 6.23 to 8.98 μg / mL, which was significantly improved compared to the PT protein concentration of 3.5 μg / mL of the wild-type strain.
[0047] [Table 4]
[0048] As can be seen from the above results, the genetically engineered bacterium OEPRN-PT obtained in the present invention significantly shortened the fermentation time, and at the end of fermentation, the expression level of the target protein in the fermentation liquid was higher, among which the expression level of the PRN protein was very high, and the concentration of the expressed PT protein was higher than that of the wild-type strain WT.
[0049] Example 4: Purification of PRN protein from genetically engineered strain OEPRN-PT 4.1 Experimental materials Bacterial lysis buffer: 10 mM Tris-HCl, 150 mM NaCl, 1 mM PMSF (benzylsulfonyl fluoride) Reconstitution buffer: 35mM NaCl, 25mM Tris-HCl Buffer A: 50mM Tris-HCl Buffer B: 50mM Tris-HCl, 1M NaCl 4.2 Experimental steps 4.2.1 Crude purification of PRN protein The genetically engineered bacteria OEPRN-PT fermentation liquid obtained in Example 3 was centrifuged at 4°C for 30 minutes to obtain a bacterial cell precipitate, which was then redissolved in 1000 mL of bacteria disruption buffer, incubated at 60°C for 1 hour, centrifuged at 4°C for 30 minutes, and the supernatant was collected to obtain a total of 950 mL of leachate. An aqueous ammonium sulfate solution was added to the leachate to precipitate for 2 hours, and the leachate was left to stand at room temperature for 1 hour, and then centrifuged at 4°C for 50 minutes to collect the precipitate.
[0050] The precipitate obtained by ammonium sulfate salting out was redissolved in 600 mL of redissolution buffer, and after redissolution overnight, centrifuged at 4° C. for 50 minutes to collect a total of 500 mL of supernatant. The supernatant was ultrafiltered with a Pellicon XL PXB10C50 ultrafiltration module to exchange for 50 mM Tris-HCl, and 260 mL of ultrafiltrate was obtained.
[0051] 4.2.2 Purification of PRN protein The QHP column was regenerated by sequentially washing with 0.5 M NaOH aqueous solution and Buffer B. It was then equilibrated with 5 column volumes of Buffer A at a flow rate of 2 mL / min. The ultrafiltrate obtained in step 4.2.1 was loaded onto the QHP column at a flow rate of 2 mL / min. After the flow-through was complete, it was subsequently washed with 5 column volumes of Buffer A.
[0052] Next, the sample was eluted with 13% (volume ratio) Buffer B, and the elution peak was collected, in which the PRN protein was present. This elution peak was named QHP-13%B (Figure 5). The eluted sample contained almost no contaminating proteins. The collected proteins were detected by the BCA method and HPLC, respectively.
[0053] 4.3 Experimental results The BCA detection results are shown in Table 5. The concentration of PRN protein changed according to the collection time, reaching a maximum of 655.5223 (μg / mL), nearly exceeding 194 μg / mL at each stage. The HPLC results (see Figure 6) showed that the concentration of PRN reached 97.52%.
[0054] [Table 5]
[0055] Example 5: Purification of PT protein obtained from genetically engineered strain OEPRN-PT 5.1 Experimental steps Test materials: Buffer C: 50mM PB (phosphate buffer) + 2M urea Buffer D: 50mM PB+1M NaCl+2M urea 5.1.1 Crude purification of PT proteins The fermentation solution of the genetically engineered bacteria OEPRN-PT obtained in Example 3 was centrifuged at 4°C for 30 minutes, and the supernatant was retained. The filtered supernatant was concentrated using a 10 kDa membrane package, and then passed through a membrane filter to remove impurities. The supernatant was then replaced with 50 mM PB by ultrafiltration, yielding a total of 300 mL of ultrafiltrate.
[0056] 5.1.2 Purification of PT proteins The SP-inspire column was regenerated by sequentially washing with 0.5M NaOH aqueous solution and Buffer D. Then, Buffer C was run at a flow rate of 2 mL / min to equilibrate to baseline. 145 mL of ultrafiltrate was loaded onto the SP-inspire column at a flow rate of 2 mL / min. After the flow-through was completed, it was subsequently washed with Buffer C until it returned to baseline, and impurities were washed with 8% (volume ratio) Buffer D.
[0057] The protein was eluted with 20% (volume ratio) of Buffer D, and the elution peak containing the PT protein was collected and named SP-20%B.
[0058] Purification of PT protein from wild-type strain WT: The fermented bacterial liquid after fermentation of the wild-type strain WT was used, and the purification steps were the same as those in 5.1.1 and 5.1.2 above.
[0059] 5.2 Experimental results The purification results of the PT protein produced by the genetically engineered bacterium OEPRN-PT and the wild-type strain WT are shown in Figures 7 and 8, respectively. As is clear from Figures 7 and 8, when the PT protein was obtained by purifying the genetically engineered bacterium OEPRN-PT using ion chromatography, the eluted sample was not contaminated with FHA protein and had a high PT content. When the PT was obtained by purifying the WT strain using ion chromatography, the eluted sample was contaminated with FHA protein and had a low PT content.
[0060] As can be seen from the above, the present invention provides a B. pertussis genetically engineered strain OEPRN-PT that overexpresses PRN protein by constructing a recombinant expression vector for blocking the expression of FHA protein and introducing it into a wild-type B. pertussis strain, which can simultaneously obtain high expression amounts of PRN protein and PT protein. The fermentation time of this strain is short, and the contents of PRN protein and PT protein are significantly higher than those of the wild strain. In addition, the two proteins are present in the bacterial cell precipitate and the bacterial liquid supernatant respectively, which is convenient for separation and purification, and meets the needs of industrial production.
[0061] Although the present invention has been described in detail above, it is obvious to those skilled in the art that various modifications and variations may be made to the present invention without departing from the spirit and scope of the present invention. The scope of the present invention is not limited to the above detailed description, but is governed by the claims.
[0062] The above is only a preferred embodiment of the present invention, and does not impose any limitations on the present invention. Although the present invention is disclosed in the above preferred embodiment, the present invention is not limited thereto. Those skilled in the art can use the technology disclosed above to make some changes or modifications to obtain equivalent embodiments with changes, without departing from the technical solution of the present invention. However, any changes or equivalent changes or modifications made to the above embodiments in accordance with the spirit of the present invention, as long as they do not deviate from the technical solution of the present invention, still belong to the scope of the technical solution of the present invention.
Claims
1. a pertactin protein expression cassette; Resistance screening genes; a filamentous hemagglutinin gene upstream recombinant nucleic acid fragment and a downstream recombinant nucleic acid fragment; A pertussis antigen recombinant expression vector, wherein the pertactin protein expression cassette is located between the upstream recombinant nucleic acid fragment and the downstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene, and the upstream recombinant nucleic acid fragment and the downstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene can undergo homologous recombination with the upstream and downstream of the filamentous hemagglutinin gene, respectively.
2. the expression vector is a plasmid pUC57, a pEASY-Blunt cloning vector, or a pBBR series vector; 2. The recombinant expression vector of claim 1, which is preferably the plasmid pUC57.
3. the upstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene comprises or is a nucleic acid fragment consisting of consecutive bases from positions m to 1000 of the nucleotide sequence shown in SEQ ID NO: 2, wherein m is a natural number of 57 or less, and preferably the upstream recombinant nucleic acid fragment of the filamentous hemagglutinin gene is a nucleic acid fragment consisting of consecutive bases from positions 57 to 1000 of the nucleotide sequence shown in SEQ ID NO: 2, Preferably, the recombinant nucleic acid fragment downstream of the filamentous hemagglutinin gene comprises or is a nucleic acid fragment consisting of consecutive bases from positions 1 to n of the nucleotide sequence shown in SEQ ID NO: 4, where n is a natural number between 798 and 1000, and more preferably, the recombinant nucleic acid fragment downstream of the filamentous hemagglutinin gene is a nucleic acid fragment consisting of consecutive bases from positions 1 to 798 of the nucleotide sequence shown in SEQ ID NO:
4.
4. The recombinant expression vector of claim 1 , wherein the nucleotide sequence of the pertactin expression cassette is set forth in SEQ ID NO:
3.
5. the resistance screening gene is one or more selected from the group consisting of a kanamycin resistance screening gene, a tetracycline resistance screening gene, an ampicillin resistance screening gene, and a chloramphenicol resistance screening gene, and is preferably a kanamycin resistance screening gene; Preferably, the nucleotide sequence of the kanamycin resistance screening gene is shown in SEQ ID NO: 5, the nucleotide sequence of the tetracycline resistance screening gene is shown in SEQ ID NO: 6, the nucleotide sequence of the ampicillin resistance screening gene is shown in SEQ ID NO: 7, and the nucleotide sequence of the chloramphenicol resistance screening gene is shown in SEQ ID NO:
8. A recombinant expression vector according to any one of claims 1 to 4.
6. A genetically engineered bacterium recombinantly expressing a pertussis antigen, comprising the recombinant expression vector of claim 1.
7. Transforming wild-type Bordetella pertussis competent cells with the recombinant expression vector of claim 1, Preferably, the wild-type Bordetella pertussis is the wild-type Bordetella pertussis strain ATCC-BAA-589; Preferably, the transformation is electrotransformation, and more preferably, the electrotransformation comprises linearizing the recombinant expression vector described in claim 1 and electrotransforming wild-type Bordetella pertussis competent cells under conditions of 2500 V and 5 milliseconds.
8. The method of claim 7, further comprising screening wild-type Bordetella pertussis competent cells after transformation using the resistance screening gene.
9. A method for producing a pertussis antigen, comprising fermenting the recombinant pertussis antigen-expressing genetically engineered bacterium of claim 6.
10. The fermentation (1) inoculating the recombinant pertussis antigen-expressing genetically engineered bacteria according to claim 6 onto a plate containing Bordet-Jung medium and culturing for 40-72 hours, preferably 48 hours; (2) inoculating the bacterial cells obtained in step (1) into a shake flask containing MSS medium and culturing for 22.5 to 25 hours; (3) inoculating the bacterial solution obtained in step (2) into a fermenter containing an MSS medium and culturing for 28 to 30 hours.
11. The method of claim 9, wherein the pertussis antigen is pertactin and / or pertussis toxin.