CN117844719A - Attenuated bordetella pertussis recombinant strain, construction method and application thereof - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides an attenuated pertussis recombinant strain, a construction method and application thereof, and belongs to the technical field of genetic detoxification of pertussis toxin genes. The recombinant strain is obtained by modifying mature S1 subunit of a wild-type CS strain by one or more amino acid mutations or S1 subunit full-length deletion with wild-type pertussis as a background; wherein the amino acid sequence of the mature S1 subunit of the wild-type CS strain is shown as SEQ ID No. 1. The recombinant strain has kanamycin resistance. Compared with the WHO generation 1 pertussis toxin standard JNIH-5, the pertussis toxin expressed by the recombinant strain has lower toxicity; the maximum growth concentration was higher compared to wild type bordetella pertussis.
Description
Technical Field
The invention belongs to the technical field of pertussis toxin gene genetic detoxification, and in particular relates to an attenuated recombinant strain of pertussis bacillus, a construction method and application thereof.
Background
Pertussis Toxin (PT) is a major virulence factor produced by bordetella Pertussis, which is a bacterial Toxin having an a-B structure consisting of S1, S2, S3, S4, S5 subunits at 1:1:1:2:1, wherein the a protomer consists of S1 subunits and the B oligomer consists of S2, S3, S4, S5. The A-protomer and the B-oligomer of pertussis toxin have different biological functions respectively. The A protomer has ADP-ribosyl transferase activity, and can catalyze transfer of adenosine diphosphate ribose to cysteine residue at C terminal of GTP-binding protein, so that GTP-binding protein can block adenylate cyclase from binding with receptor, and increase of intracellular cAMP (cyclic adenosine monophosphate) can generate various biological effects such as histamine sensitization, leucocytosis, insulin secretion and the like. The B oligomer is a nontoxic oligomer, binds to various receptors on the surface of eukaryotic cells, and mediates endocytosis of toxic S1 subunits to target proteins in the cells. The interaction of ATP (adenosine triphosphate) with the B oligomer can lead to the transfer of the S1 subunit to the cytoplasmic matrix and release in the endoplasmic reticulum, while mediating holotoxin separation before translocation occurs on the cell membrane. Studies have shown that B oligomers of pertussis toxin can activate and modulate immune responses as immunopotentiators.
Pertussis toxin is the major component of currently available vaccines, and the presence of antibodies to this toxin is associated with protecting children from pertussis disease. Because pertussis toxin has a plurality of biological activities, after the nano-gram level non-detoxified PT is injected into experimental animals, the pertussis toxin can cause the reactions of peripheral blood leucocyte increase, histamine sensitization, anaphylaxis enhancement of vascular active substances and the like. Thus, pertussis toxin must be detoxified before use in vaccine preparation.
The current modes of detoxification of PT are mainly divided into chemical detoxification and genetic detoxification. The detoxicant used for chemical detoxication is mainly formaldehyde and glutaraldehyde, and hydrogen peroxide is also used for detoxication. Genetic detoxification is the introduction of mutants into PT by genetic engineering methods, which typically result in point mutations in the S1 subunit of PT, thereby greatly reducing toxicity but retaining its immunogenicity. Because the overall conformation of PT protein is changed by chemically detoxified PT, the serological antibody level is influenced, and the currently developed vaccine can not continuously and effectively control diseases for a long time, the development of a genetic detoxication method is necessary, and a foundation is laid for the research and development of a novel pertussis vaccine.
Disclosure of Invention
The invention provides an attenuated recombinant strain of bordetella pertussis. The recombinant strain is obtained by modifying a wild pertussis CS strain as a background, has kanamycin resistance, and has lower toxicity compared with a WHO 1 st generation pertussis toxin standard product JNIH-5.
The existing recombinant strain of genetic detoxified PT is commonly obtained by mutation of PT-S1 subunit R9K/E129G of European and American pertussis Tohama strain, and also is obtained by gene editing of bordetella parapertussis and bordetella bronchiseptica. The wild type pertussis is derived from a CS strain for producing Chinese pertussis vaccine, wherein PT-S1 subunit is naturally different from Tohama strain at 194 amino acid (methionine is at 194 of the CS strain, and isoleucine is at 194 of the Tohama strain). Thus, even if mutation (e.g., R9K or E129G) at the same site is performed on the CS strain, the effect is not exactly the same as that of the Tohama strain. In view of the uniqueness of CS strains, the construction of genetically detoxified recombinant strains thereof has important significance.
Specifically, the invention adopts the following technical scheme to realize the aim:
the pertussis recombinant strain is obtained by mutating one or more amino acids of a pertussis toxin mature S1 subunit of a wild-type pertussis CS strain or deleting the whole length of the S1 subunit; the recombinant bordetella pertussis strain has kanamycin resistance; the amino acid sequence of the mature S1 subunit of pertussis toxin of the wild type pertussis bacillus CS strain is shown as SEQ ID No. 1.
In a preferred embodiment, the pertussis recombinant strain expresses a pertussis toxin mature S1 subunit comprising the double site mutations R9K and E129G in the amino acid sequence shown in SEQ ID No. 1.
In a further preferred embodiment, the amino acid sequence of the mature S1 subunit of pertussis toxin expressed by the recombinant strain of pertussis is depicted as SEQ ID No. 2.
In a still further preferred embodiment, the recombinant bordetella pertussis strain comprises in its genome the nucleotide sequence shown in SEQ ID No.3 or comprises a nucleotide sequence having more than 95% homology with the amino acid sequence shown in SEQ ID No.2 after translation.
The invention also provides a construction method of the pertussis recombinant strain, which comprises the following steps: constructing a recombinant plasmid pB-Kana-gS1 containing PT-S1 subunit mutant genes, and preparing competent cells of wild type pertussis CS strain; preparing the recombinant plasmid pB-Kana-gS1 into a linearized DNA recombinant fragment containing PT-S1 subunit mutant genes, electrically transferring the linearized DNA recombinant fragment containing the PT-S1 subunit mutant genes into the CS strain competent cells, resuscitating, and screening kanamycin resistance to obtain positive strains, namely the recombinant strain of the pertussis bacillus with PT-S1 subunit gene mutation;
or preparing competent cells of wild pertussis CS strain, electrotransferring the pKD46 plasmid into the competent cells of the CS strain to obtain the pertussis CS strain containing the pKD46 plasmid, and preparing the competent cells containing the pKD46 plasmid; constructing a recombinant plasmid pB-delta S1 with deletion of PT-S1 subunit gene; preparing the recombinant plasmid pB-delta S1 into a linearization DNA recombinant fragment without PT-S1 subunit gene, electrically transferring the linearization DNA recombinant fragment without PT-S1 subunit gene into competent cells containing the pKD46 plasmid, resuscitating, and screening by kanamycin resistance to obtain a positive strain which is the recombinant strain of the pertussis bacillus with the PT-S1 subunit gene deleted.
In a preferred embodiment, the construction method of the recombinant plasmid pB-Kana-gS1 comprises the following steps: the pBluescript II SK (+) plasmid is taken as a vector skeleton, restriction enzyme sites XhoI and BamHI are taken as target fragment connecting sites, and a 5 'homology arm of a PT-S1 subunit gene, a NOS promoter, a Kana resistance gene, a PT-S1 subunit mutant gene and a 3' homology arm of the PT-S1 subunit gene are integrated into the pBluescript II SK (+) plasmid through gene synthesis.
In a preferred embodiment, the construction method of the recombinant plasmid pB-DeltaS 1 comprises the steps of: the vector skeleton is pBluescript II SK (+) plasmid, the restriction enzyme sites XhoI and BamHI are used as target fragment connecting sites, and the 5 'homology arm of PT-S1 subunit gene, the Amp promoter, the Kana resistance gene and the 3' homology arm of PT-S1 subunit gene are integrated into the pBluescript II SK (+) plasmid through gene synthesis.
In a preferred embodiment, the method for preparing competent cells of wild-type bordetella pertussis comprises the steps of: inoculating wild pertussis to Stainer-Scholte liquid culture medium, and culturing to OD 600nm After bacterial cells are collected and washed with sterile double distilled water precooled to 2-8 ℃, the bacterial cells are washed 2 times with 10% (V/V) glycerol precooled to 2-8 ℃ and then sub-packaged with 100 μl per tube, and stored at-80 ℃.
In a further preferred embodiment, the final concentration of bacteria in the SSM medium after inoculation is controlled to be 2 hundred million CFU/mL.
In a preferred embodiment, 80 to 120. Mu.L of the competent cells are mixed with 1 to 4. Mu.g of the linearized DNA recombinant fragment at the time of electrotransformation.
In a preferred embodiment, the conditions of the electrical transfer are: the electrode spacing of the electric shock cup is 1mm, the capacitance is 25 mu F, the resistance is 200 omega, and the voltage is 2400V.
In a preferred embodiment, the recovery means that the bacterial liquid obtained after electrotransformation is added into SSM culture medium preheated at 37 ℃, mixed uniformly and cultured for 12-16 hours under the conditions of 35+/-2 ℃ and 220 rpm.
In a preferred embodiment, the step of screening for kanamycin resistance comprises: the resuscitated bacterial solution was spread on a 15% sheep blood Borset-Gengou resistant plate containing kanamycin and incubated at 35.+ -. 2 ℃ for 3-5 days.
Compared with the WHO 1 st generation pertussis toxin standard product JNIH-5, the pertussis recombinant strain constructed in the invention has lower toxicity of the expressed pertussis toxin, and can be used for preparing products for treating or/and detecting or/and preventing pertussis diseases. The product for preventing pertussis disease is preferably a pertussis vaccine.
The invention has the following beneficial effects: (1) The pertussis recombinant strain is constructed by adopting electric transduction to introduce linear DNA recombinant fragments so as to finish the editing and transformation of target genes. Compared with the traditional method of introducing recombinant plasmid into pertussis bacillus by using escherichia coli in a combination way, the nucleic acid introduction way in the invention avoids the introduction and pollution of escherichia coli, meanwhile, the introduced nucleic acid is linear DNA, can not be accumulated and copied in thalli, gradually disappears along with the metabolism and passage of the strain after non-recombination or recombination, and can not pollute genetic background. (2) The pertussis recombinant strain prepared in the invention has kanamycin resistance and is convenient for recombinant screening; the recombinant strain grew at a higher maximum concentration of bacteria than the wild type pertussis. In the CHO cell agglutination assay, the result obtained by quantitative detection by the PT multi-antibody ELISA double sandwich method shows that the secreted PT protein has lower toxicity compared with the WHO generation 1 pertussis toxin standard product JNIH-5. Wherein, when the mature S1 subunit (PT-S1) of pertussis toxin contains an R9K single mutation site, the PT toxicity is reduced to 0.0081 percent; when the mature S1 subunit (PT-S1) of pertussis toxin contains double site mutations of R9K and E129G, PT toxicity is reduced to 0.0016%. When the whole length of the S1 subunit of pertussis toxin is deleted, the PT multi-antibody-ELISA double-antibody sandwich method can not detect the PT protein content in culture supernatant, and the S1 subunit monoclonal antibody 1B7 can not detect the band in immunoblotting detection. The recombinant strain can lay a foundation for the research of the action mechanism of the PT protein of the pertussis CS strain and the research of the gene detoxified pertussis vaccine.
Drawings
FIG. 1 is a schematic diagram of the structures of pB-Kana-gS1 plasmid, pB-DeltaS 1 plasmid and linearized DNA recombinant fragment;
FIG. 2 is a graph showing the growth of recombinant strains 18123, FE3, FE16 and the B.pertussis CS strain (WT);
FIG. 3 is a graph showing the aging of PT proteins secreted by recombinant strains 18123, FE3 and FE 16;
FIG. 4 is a graphical representation of microscopic examination of CHO cell agglutination for recombinant strains FE3, FE16 and pertussis CS strain (WT) and a statistical bar graph of minimum PT content required for agglutination;
FIG. 5 is a diagram showing the results of immunoblotting analysis of 1B7 mAb against PT proteins of recombinant strains 18123, FE3 and FE16.
Detailed Description
The following description sets forth a clear and complete description of the present invention, in connection with embodiments, so that those skilled in the art will fully understand the present invention. It will be apparent that the described embodiments are only some, but not all, of the preferred embodiments of the invention. Any equivalent alterations or substitutions for the following embodiments without any inventive effort by those of ordinary skill in the art are intended to be within the scope of the present invention.
Ordinal terms such as "first," "second," and the like, as used herein, are used solely for descriptive purposes to distinguish between similar objects and are not to be construed as indicating or implying a relative importance or an implicit indication of the number of technical features indicated. The methods not described in detail in the examples below are all conventional methods well known to those skilled in the art. The bordetella pertussis CS strain used in the examples below was deposited under accession number CMCC 58003.CHO cells were supplied by all of the company responsible for the study of the biological products of martial arts. Stainer-Scholte liquid Medium (SSM) (Shandong Tuo Pu bioengineering Co., ltd., product number: M2343) contains the following main components: every 1000mL contains 10.72g L-sodium glutamate, 0.24g L-proline, 1.52g aminomethane, 2.5g sodium chloride, 0.2g potassium chloride, 0.1g magnesium chloride hexahydrate, 0.02g calcium chloride, 0.5g potassium dihydrogen phosphate, 0.02g ascorbic acid, 0.004g nicotinic acid, 0.1g glutathione, 0.01g ferrous sulfate heptahydrate, and pH=7.6+/-0.2. The Borset-Gengou (BG) medium (Guangdong Cryptographic microorganism Co., ltd., cat# 027504) contained the following main components: each 1000mL contains 4.5g potato starch, 20g peptone, 5.5g sodium chloride, 14g agar, and pH=6.7+ -0.2. CHO cell culture medium DMEM/F12 (1:1) (cat# 11330032) was purchased from Gibco corporation.
R9K represents the mutation of Arginine (abbreviated as R) at position 9 on the mature S1 subunit of pertussis toxin to Lysine (abbreviated as K), E129G represents the mutation of glutamic acid (abbreviated as E) at position 129 on the mature S1 subunit of pertussis toxin to Glycine (abbreviated as G).
Example 1
This example provides a method for preparing a recombinant strain of PT-S1 mutated bordetella pertussis comprising the steps of:
1.1 preparation of competent cells
50mL of SSM medium was added to a 250mL shake flask, and the wild type pertussis CS strain was inoculated onto the medium at a final concentration of 2 hundred million CFU/mL. Placing the shake flask in a constant temperature shaking table, culturing at 35deg.C and 220rpm to OD 600nm ＝0.5～0.7。
In a biosafety cabinet, the bacterial liquid was collected by centrifugation at 4000g at 4℃for 15 minutes using a 50mL sterile centrifuge tube, and the first bacterial cells were collected.
The first cell was resuspended and washed with 25mL of sterile double distilled water pre-chilled to 2-8deg.C, centrifuged at 4℃and 4000g for 15 min, and the second cell was collected.
The second cell was resuspended in 10mL of 10% (V/V) glycerol precooled to 2-8℃and placed in ice water for 10 min, centrifuged at 4000g at 4℃for 15 min, and the third cell was collected.
The third cell was resuspended in 5mL of 10% (V/V) glycerol precooled to 2-8℃and placed in ice water for 10 min, centrifuged at 4000g at 4℃for 15 min, and the fourth cell was collected.
Re-suspending the fourth thallus with 1mL 10% (V/V) glycerol pre-cooled to 2-8deg.C, sub-packaging with 100 μL/tube into sub-packaging tubes pre-cooled to 2-8deg.C, and storing at-80deg.C.
1.2 preparation of linearized recombinant DNA fragments
The pBluescript II SK (+) plasmid is taken as a vector skeleton, restriction enzyme sites XhoI and BamHI are taken as target fragment connecting sites, and recombinant plasmids pB-Kana-gS1 (shown in figure 1) sequentially containing a 5 'homology arm (511 bp), a NOS promoter (180 bp), a Kana resistance gene (NeoR/KanR, 795 bp), a PT-S1 subunit R9K/E129G mutant gene (comprising a leader peptide nucleotide sequence of 102bp and 810 bp) and a 3' homology arm (391 bp) are synthesized through genes (the gene synthesis work is completed by biological engineering (Shanghai).
PCR amplification was performed using plasmid pB-Kana-gS1 (nucleotide sequence shown as SEQ ID No. 4) as a template and primer F1 (atcccgaattcgtcgcctc) and primer R1 (tggtcatgatgcagaacgtcg) as upstream and downstream primers, respectively. And (3) carrying out 1% agarose gel electrophoresis and gel cutting recovery and purification on the obtained PCR amplification product to finally obtain the linearized DNA recombinant fragment with the nucleotide sequence shown as SEQ ID No. 3. Wherein, the reaction system of PCR amplification is: 4. Mu.L of the upstream primer (10. Mu. Mol/L), 4. Mu.L of the downstream primer (10. Mu. Mol/L), 50ng of plasmid pB-Kana-gS1 as a template, 25. Mu.L of 2X Phanta Max Master Mix (purchased from Norpran Corp.) (cat. No.: P525) and ddH were used 2 O was made up to a total volume of the reaction system of 50. Mu.L. The PCR reaction procedure was as follows: in the first stage, 94 ℃ for 3 minutes, and the cycle number is 1; second stage, 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃,3 minutes, cycle number 35; in the third stage, the temperature is 72 ℃ and the cycle number is 1 for 10 minutes.
Amino acid sequence of mature S1 subunit of pertussis toxin of wild type bordetella pertussis CS strain SEQ ID No.1: DPPATVYRYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFYGAASSYFEYVDTYGDNAGRILAGALATYQSEYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRMAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSF.
The amino acid sequence of mature S1 subunit of pertussis toxin expressed by recombinant strain of pertussis bacillus SEQ ID No.2: DPPATVYKYDSRPPEDVFQNGFTAWGNNDNVLDHLTGRSCQVGSSNSAFVSTSSSRRYTEVYLEHRMQEAVEAERAGRGTGHFIGYIYEVRADNNFYGAASSYFEYVDTYGDNAGRILAGALATYQSGYLAHRRIPPENIRRVTRVYHNGITGETTTTEYSNARYVSQQTRANPNPYTSRRSVASIVGTLVRMAPVIGACMARQAESSEAMAAWSERAGEAMVLVYYESIAYSF.
Sequence of linearized DNA recombination fragment comprising R9K/E129G mutation SEQ ID No.3: atcccgaattcgtcgcctcgccctggttcgccgtcatggcccccaagggaaccgaccccaagataatcgtcctgctcaaccgccacatcaacgaggcgctgcagtccaaggcggtcgtcgaggcctttgccgcccaaggcgccacgccggtcatcgccacgccggatcagacccgcggcttcatcgcagacgagatccagcgctgggccggcgtcgtgcgcgaaaccggcgccaagctgaagtagcagcgcagccctccaacgcgccatccccgtccggccggcaccatcccgcatacgtgttggcaaccgccaacgcgcatgcgtgcagattcgtcgtacaaaaccctcgattcttccgtacatcccgctactgcaatccaacacggcatgaacgctccttcggcgcaaagtcgcgcgatggtaccggtcaccgtccggaccgtgctgacccccctgccatggtgtgatccgtaaaataggcaccatcaaaacgcagaggggaagacggggaaccgcaacgttgaaggagccactgagccgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgttcaaaagtcgcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgttccataaattcccctcggtatccaattagagtctcatattcaccctcaactcgatcgaggctcggggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactccaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcggatgcccgacggcgaggatctcgtcgtgacccacggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagacgggatgcgttgcactcgggcaattcgccaaaccgcaagaacaggctggctgacgtggctggcgattcttgccgtcacggcgcccgtgacttcgccggcatgggccgacgatcctcccgccaccgtatacaagtatgactcccgcccgccggaggacgttttccagaacggattcacggcgtggggaaacaacgacaatgtgctcgaccatctgaccggacgttcctgccaggtcggcagcagcaacagcgctttcgtctccaccagcagcagccggcgctataccgaggtctatctcgaacatcgcatgcaggaagcggtcgaggccgaacgcgccggcaggggcaccggccacttcatcggctacatctacgaagtccgcgccgacaacaatttctacggcgccgccagctcgtacttcgaatacgtcgacacttatggcgacaatgccggccgtatcctcgccggcgcgctggccacctaccagagcgggtatctggcacaccggcgcattccgcccgaaaacatccgcagggtaacgcgggtctatcacaacggcatcaccggcgagaccacgaccacggagtattccaacgctcgctacgtcagccagcagactcgcgccaatcccaacccctacacatcgcgaaggtccgtagcgtcgatcgtcggcacattggtgcgcatggcgccggtgataggcgcttgcatggcgcggcaggccgaaagctccgaggccatggcagcctggtccgaacgcgccggcgaggcgatggttctcgtgtactacgaaagcatcgcgtattcgttctagacctggcccagccccgcccaactccggtaattgaacagcatgccgatcgaccgcaagacgctctgccatctcctgtccgttctgccgttggccctcctcggatctcacgtggcgcgggcctccacgccaggcatcgtcattccgccgcaggaacagattacccagcatggcggcccctatggacgctgcgcgaacaagacccgtgccctgaccgtggcggaattgcgcggcagcggcgatctgcaggagtacctgcgtcatgtgacgcgcggctggtcaatatttgcgctctacgatggcacctatctcggcggcgaatatggcggcgtgatcaaggacggaacacccggcggcgcattcgacctgaaaacgacgttctgcatcatgacca.
Nucleotide sequence of plasmid pB-Kana-gS1 SEQ ID No.4: ctaaattgtaagcgttaatattttgtta aaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaag
gaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccaca
cccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcg
gtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccaggg
ttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccg
ggccccccctcgagatcccgaattcgtcgcctcgccctggttcgccgtcatggcccccaagggaaccgaccccaagat
aatcgtcctgctcaaccgccacatcaacgaggcgctgcagtccaaggcggtcgtcgaggcctttgccgcccaaggcg
ccacgccggtcatcgccacgccggatcagacccgcggcttcatcgcagacgagatccagcgctgggccggcgtcgt
gcgcgaaaccggcgccaagctgaagtagcagcgcagccctccaacgcgccatccccgtccggccggcaccatccc
gcatacgtgttggcaaccgccaacgcgcatgcgtgcagattcgtcgtacaaaaccctcgattcttccgtacatcccgcta
ctgcaatccaacacggcatgaacgctccttcggcgcaaagtcgcgcgatggtaccggtcaccgtccggaccgtgctga
cccccctgccatggtgtgatccgtaaaataggcaccatcaaaacgcagaggggaagacggggaaccgcaacgttgaa
ggagccactgagccgcgggtttctggagtttaatgagctaagcacatacgtcagaaaccattattgcgcgttcaaaagtc
gcctaaggtcactatcagctagcaaatatttcttgtcaaaaatgctccactgacgttccataaattcccctcggtatccaatta
gagtctcatattcaccctcaactcgatcgaggctcggggaagccctgcaaagtaaactggatggctttcttgccgccaag
gatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattg
cacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatg
ccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactc
caagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaa
gcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaa
gtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaac
atcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggc
tcgcgccagccgaactgttcgccaggctcaaggcgcggatgcccgacggcgaggatctcgtcgtgacccacggcgat
gcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccg
ctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgcttta
cggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagacgggatgcgttgcact
cgggcaattcgccaaaccgcaagaacaggctggctgacgtggctggcgattcttgccgtcacggcgcccgtgacttcg
ccggcatgggccgacgatcctcccgccaccgtatacaagtatgactcccgcccgccggaggacgttttccagaacgga
ttcacggcgtggggaaacaacgacaatgtgctcgaccatctgaccggacgttcctgccaggtcggcagcagcaacag
cgctttcgtctccaccagcagcagccggcgctataccgaggtctatctcgaacatcgcatgcaggaagcggtcgaggc
cgaacgcgccggcaggggcaccggccacttcatcggctacatctacgaagtccgcgccgacaacaatttctacggcg
ccgccagctcgtacttcgaatacgtcgacacttatggcgacaatgccggccgtatcctcgccggcgcgctggccaccta
ccagagcgggtatctggcacaccggcgcattccgcccgaaaacatccgcagggtaacgcgggtctatcacaacggca
tcaccggcgagaccacgaccacggagtattccaacgctcgctacgtcagccagcagactcgcgccaatcccaacccc
tacacatcgcgaaggtccgtagcgtcgatcgtcggcacattggtgcgcatggcgccggtgataggcgcttgcatggcg
cggcaggccgaaagctccgaggccatggcagcctggtccgaacgcgccggcgaggcgatggttctcgtgtactacg
aaagcatcgcgtattcgttctagacctggcccagccccgcccaactccggtaattgaacagcatgccgatcgaccgcaa
gacgctctgccatctcctgtccgttctgccgttggccctcctcggatctcacgtggcgcgggcctccacgccaggcatcg
tcattccgccgcaggaacagattacccagcatggcggcccctatggacgctgcgcgaacaagacccgtgccctgacc
gtggcggaattgcgcggcagcggcgatctgcaggagtacctgcgtcatgtgacgcgcggctggtcaatatttgcgctct
acgatggcacctatctcggcggcgaatatggcggcgtgatcaaggacggaacacccggcggcgcattcgacctgaaa
acgacgttctgcatcatgaccaggatccactagttctagagcggccgccaccgcggtggagctccagcttttgttcccttt
agtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacac
aacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctc
actgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggttt
gcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctc
actcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaa
aaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc
gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc
gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctc
acgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgacc
gctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggt
aacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaa
ggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaa
ccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttga
tcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttc
acctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgctt
aatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacga
tacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac。
1.3, electrotransformation and plate screening
Competent cells of the bordetella pertussis CS strain prepared in step 1.1 were removed, and slowly thawed by standing on ice for 5 min. In a biosafety cabinet, 2. Mu.g of the linearized DNA recombinant fragment prepared in step 1.2 was added to 100. Mu.L of competent cells, gently mixed, and incubated on ice for 5 minutes.
Taking out the mixture of competent cells and linearized DNA recombinant fragments, placing in a precooled 1mm specification electric shock cup, and standing on ice for 10 min.
The electric conversion conditions are set as follows: the electric shock cup has an electrode spacing of 1mm, a capacitance of 25 μF, a resistance of 200Ω and a voltage of 2400V. Taking the electric shock cup to wipe the outer surface for condensing water, putting the electric shock cup in an electric shock tank for clicking, taking out the electric shock cup after receiving the indication sound, adding 500mL of SSM culture medium preheated at 37 ℃ within 1 minute, and blowing and uniformly mixing the electric shock cup.
The bacterial solution was transferred to a 50mL sterile centrifuge tube, SSM medium preheated at 37℃was added to a volume of 3mL, and resuscitated on a constant temperature shaking table at 35.+ -. 2℃and 220rpm for 12 to 16 hours.
Subsequently, the bacterial solution was plated on a 15% sheep blood BG resistance plate containing 25. Mu.g/mL kanamycin (Kana), and cultured at 35.+ -. 2 ℃ for 3-5 days, and the strain positive for kanamycin resistance was selected.
1.4, genome level detection of recombinant strains
Pertussis bacterial colonies obtained by culture on a Kana resistant plate are picked, amplified and cultured in SSM medium containing 25 mug/mL Kana, and a proper amount of bacterial liquid is collected to extract genome.
The extracted genome was amplified by PCR using the primer F2 (cgtttcgcatgattgaacaagatg) and the primer R2 (ggtaagggcgtaagtctcgaacg) as the upstream and downstream primers, respectively. The PCR amplification reaction system is as follows: 0.8. Mu.L of the upstream primer (10. Mu. Mol/L), 0.8. Mu.L of the downstream primer (10. Mu. Mol/L), 50ng of plasmid pB-Kana-gS1 as a template, 5. Mu.L of 2X Phanta Max Master Mix (purchased from Nuo vozan Co., ltd., cat# P525) and ddH were used 2 O is added to the total volume of the reaction system to be 10 mu L; the amplification procedure was: in the first stage, 94 ℃ for 3 minutes, and the cycle number is 1; second stage, 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃,3 minutes, cycle number 35; in the third stage, the temperature is 72 ℃ and the cycle number is 1 for 10 minutes.
The PCR amplified product adopts a first detection primer (gttgcactcgggcaattc) to carry out sequence detection, and the pertussis toxin S1 subunit is subjected to comparison and confirmation to obtain a pertussis bacillus recombinant strain with R9K/E129G double-site mutation, which is named as FE3; a recombinant strain of pertussis having a R9K single point mutation in the S1 subunit of pertussis toxin was obtained and designated FE16.
In step 1.3, the amount of competent cells was 80 to 120. Mu.L, and the amount of linearized DNA recombinant fragment was 1 to 4. Mu.g, which did not affect the experimental results. There is no sequence between step 1.1 and step 1.2.
Example 2
This example provides a method for preparing recombinant strains of bordetella pertussis having a deletion of the PT-S1 subunit, comprising the steps of:
2.1 preparation of competent cells of the B.pertussis CS strain according to the same method as in step 1.1 of example 1.
2.2, electrotransport pKD46 plasmid and resistance selection
The pKD46 plasmid (ampicillin resistance, ampR) contained the Red recombination system, and the pkD plasmid was introduced to increase the efficiency of deletion recombination. Competent cells of the bordetella pertussis CS strain prepared in step 2.1 were removed, and slowly thawed by standing on ice for 5 min. In a biosafety cabinet, 1 μg of pKD46 plasmid was added to 100 μl of competent cells, gently mixed, and incubated on ice for 5 min.
The mixture of competent cells and pKD46 plasmid was removed and placed in a pre-chilled 1 mm-sized cuvette and allowed to stand on ice for 10 minutes. Then, the electric power was turned in the same manner as in step 1.3 of example 1.
Then the bacterial liquid is coated on a 15% sheep blood BG resistance plate containing 50 mug/mL of ampicillin, and cultured for 3-5 days at 35+/-2 ℃, and positive strains are screened out, namely the pertussis CS strain containing pKD46 plasmid.
2.3 preparation of competent cells of the bordetella pertussis CS strain containing the pKD46 plasmid
In a 250mL shake flask, the Bordetella pertussis CS strain containing the pKD46 plasmid was inoculated into 50mL SSM medium containing 50. Mu.g/mL Amp at a final inoculum concentration of 2 hundred million CFU/mL. Placing the shake flask in a constant temperature shaking table, culturing at 35deg.C and 220rpm to OD 600nm ＝0.5～0.7。
In a biosafety cabinet, the bacterial liquid was collected by a 50mL sterile centrifuge tube, centrifuged at 4000g for 15 minutes at 4℃and the fifth bacterial cell was collected.
The fifth cell was resuspended and washed with 25mL of sterile double distilled water pre-chilled to 2-8deg.C, centrifuged at 4℃and 4000g for 15 minutes, and the sixth cell was collected.
The sixth cell was resuspended in 10mL of 10% (V/V) glycerol precooled to 2-8℃and placed in ice water for 10 minutes, centrifuged at 4000g at 4℃for 15 minutes, and the seventh cell was collected.
The seventh cell was resuspended in 5mL of 10% (V/V) glycerol precooled to 2-8℃and placed in ice water for 10 minutes, centrifuged at 4000g at 4℃for 15 minutes, and the eighth cell was collected.
Re-suspending the eighth thallus with 1mL 10% (V/V) glycerol pre-cooled to 2-8deg.C, sub-packaging with 100 μL/tube into sub-packaging tubes pre-cooled to 2-8deg.C, and storing at-80deg.C.
2.4 preparation of linearized recombinant DNA fragments
The plasmid pBluescript II SK (+) is used as a vector skeleton, restriction enzyme sites XhoI and BamHI are used as target fragment connecting sites, and the 5 'homology arm (511 bp), the Amp promoter (105 bp), the Kana resistance gene (KanR, 816 bp) and the 3' homology arm (391 bp) of the PT-S1 subunit gene are integrated into the plasmid pBluescript II SK (+) by gene synthesis (order from Shanghai) to obtain a recombinant plasmid pB-DeltaS 1 (shown in figure 1).
PCR amplification was performed using plasmid pB- ΔS1 (nucleotide sequence shown as SEQ ID No. 5) as a template and primer F1 (atcccgaattcgtcgcctc) and primer R1 (tggtcatgatgcagaacgtcg) as upstream and downstream primers, respectively. And (3) carrying out 1% agarose gel electrophoresis and gel cutting recovery and purification on the obtained PCR amplification product to finally obtain the linearized DNA recombinant fragment with the nucleotide sequence shown as SEQ ID No. 6. Wherein, the reaction system of PCR amplification is: 4. Mu.L of the upstream primer (10. Mu. Mol/L), 4. Mu.L of the downstream primer (10. Mu. Mol/L), 50ng of plasmid pB-. DELTA.S1 as a template, 25. Mu.L of 2X Phanta Max Master Mix (purchased from Noruzan Corp., cat. Number: P525) and ddH were used 2 O was made up to a total volume of the reaction system of 50. Mu.L. The PCR reaction procedure was as follows: in the first stage, 94 ℃ for 3 minutes, and the cycle number is 1; second stage, 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃,3 minutes, cycle number 35; in the third stage, the temperature is 72 ℃ and the cycle number is 1 for 10 minutes.
Nucleotide sequence of plasmid pB- ΔS1 SEQ ID No.5: tggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaa
aaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccag
aaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcgg
taagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatc
ccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtca
cagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggc
caacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcc
ttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaa
caacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggata
aagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggg
tctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcagg
caactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt
actcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaa
aatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttct
gcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactc
tttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccactt
caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtg
tcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacaca
gcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccg
aagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggg
ggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggg
gcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttc
ctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccg
agcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgat
tcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctc
actcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacac
aggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctccaccg
cggtggcggccgctctagaactagtggatccatcccgaattcgtcgcctcgccctggttcgccgtcatggcccccaagg
gaaccgaccccaagataatcgtcctgctcaaccgccacatcaacgaggcgctgcagtccaaggcggtcgtcgaggcc
tttgccgcccaaggcgccacgccggtcatcgccacgccggatcagacccgcggcttcatcgcagacgagatccagcg
ctgggccggcgtcgtgcgcgaaaccggcgccaagctgaagtagcagcgcagccctccaacgcgccatccccgtccg
gccggcaccatcccgcatacgtgttggcaaccgccaacgcgcatgcgtgcagattcgtcgtacaaaaccctcgattctt
ccgtacatcccgctactgcaatccaacacggcatgaacgctccttcggcgcaaagtcgcgcgatggtaccggtcaccgt
ccggaccgtgctgacccccctgccatggtgtgatccgtaaaataggcaccatcaaaacgcagaggggaagacgggc
gcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaata
atattgaaaaaggaagagtatgagccatattcaacgggaaacgtcttgctctaggccgcgattaaattccaacatggatg
ctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccg
atgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactgg
ctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccc
cgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcg
ccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaa
taacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataa
acttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaata
ggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagtttt
ctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgat
gagtttttctaaacctggcccagccccgcccaactccggtaattgaacagcatgccgatcgaccgcaagacgctctgcc
atctcctgtccgttctgccgttggccctcctcggatctcacgtggcgcgggcctccacgccaggcatcgtcattccgccg
caggaacagattacccagcatggcggcccctatggacgctgcgcgaacaagacccgtgccctgaccgtggcggaatt
gcgcggcagcggcgatctgcaggagtacctgcgtcatgtgacgcgcggctggtcaatatttgcgctctacgatggcac
ctatctcggcggcgaatatggcggcgtgatcaaggacggaacacccggcggcgcattcgacctgaaaacgacgttct
gcatcatgaccagaattcgatatcaagcttatcgataccgtcgacctcgagggggggcccggtacccaattcgccctata
gtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatc
gccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcg
cagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtg
accgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccg
tcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggt
gatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggact
cttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttag。
Sequence of linearized DNA recombinant fragment with deletion of PT-S1 subunit gene SEQ ID No.6: atccc gaattcgtcgcctcgccctggttcgccgtcatggcccccaagggaaccgaccccaagataatcgtcctgctcaaccgccacatcaacgaggcgctgcagtccaaggcggtcgtcgaggcctttgccgcccaaggcgccacgccggtcatcgccacgccggatcagacccgcggcttcatcgcagacgagatccagcgctgggccggcgtcgtgcgcgaaaccggcgccaagctgaagtagcagcgcagccctccaacgcgccatccccgtccggccggcaccatcccgcatacgtgttggcaaccgccaacgcgcatgcgtgcagattcgtcgtacaaaaccctcgattcttccgtacatcccgctactgcaatccaacacggcatgaacgctccttcggcgcaaagtcgcgcgatggtaccggtcaccgtccggaccgtgctgacccccctgccatggtgtgatccgtaaaataggcaccatcaaaacgcagaggggaagacgggcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagccatattcaacgggaaacgtcttgctctaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattt tatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaaacctggcccagccccgcccaactccggtaattgaacagcatgccgatcgaccgcaagacgctctgccatctcctgtccgttctgccgttggccctcctcggatctcacgtggcgcgggcctccacgccaggcatcgtcattccgccgcaggaacagattacccagcatggcggcccctatggacgctgcgcgaacaagacccgtgccctgaccgtggcggaattgcgcggcagcggcgatctgcaggagtacctgcgtcatgtgacgcgcggctggtcaatatttgcgctctacgatggcacctatctcggcggcgaatatggcggcgtgatcaaggacggaacacccggcggcgcattcgacctgaaaacgacgttctgcatcatgacca.
2.5, electrotransformation and plate screening
Competent cells of the bordetella pertussis CS strain containing the pKD46 plasmid prepared in step 2.3 were removed, and left on ice for 5 min to slowly thaw. In a biosafety cabinet, 2. Mu.g of the linearized DNA recombinant fragment prepared in step 2.4 was added to 100. Mu.L of competent cells, gently mixed, and incubated on ice for 5 minutes.
Taking out the mixture of competent cells and linearized DNA recombinant fragments, placing in a precooled 1mm specification electric shock cup, and standing on ice for 10 min.
The electric conversion conditions are set as follows: the electric shock cup has an electrode spacing of 1mm, a capacitance of 25 μF, a resistance of 200Ω and a voltage of 2400V. Taking the electric shock cup to wipe the outer surface for condensing water, putting the electric shock cup in an electric shock tank for clicking, taking out the electric shock cup after receiving the indication sound, adding 500mL of SSM culture medium preheated at 37 ℃ within 1 minute, and blowing and uniformly mixing the electric shock cup.
The bacterial solution was transferred to a 50mL sterile centrifuge tube, SSM medium preheated at 37℃was added to a volume of 3mL, and ampicillin was added at a final concentration of 50. Mu.g/mL and resuscitated on a thermostatically controlled shaking table at 35.+ -. 2℃and 220rpm for 12-16 hours.
Subsequently, the bacterial solution was spread on a 15% sheep blood BG resistance plate containing 25. Mu.g/mL kanamycin, and cultured at 35.+ -. 2 ℃ for 3-5 days, and the kanamycin resistance positive strain was selected.
2.6, genome level detection of recombinant strains
Pertussis bacterial colonies obtained by culture on a Kana resistant plate are picked, amplified and cultured in SSM medium containing 25 mug/mL Kana, and a proper amount of bacterial liquid is collected to extract genome.
The primer F3 (gggaaacgtcttgctctag) and the primer R2 are respectively used as an upstream primer and a downstream primer for PCR amplification of the extracted genome. The PCR amplification reaction system is as follows: 0.8. Mu.L of upstream primer (10. Mu. Mol/L), 0.8. Mu.LA downstream primer (10. Mu. Mol/L), 50ng of plasmid pB-Kana-gS1 as a template, 5. Mu.L of 2X Phanta Max Master Mix (purchased from Novozan Co., ltd.; cat.: P525) was used as a template 2 O is added to the total volume of the reaction system to be 10 mu L; the amplification procedure was: in the first stage, 94 ℃ for 3 minutes, and the cycle number is 1; second stage, 94 ℃, 30 seconds, 58 ℃, 30 seconds, 72 ℃,3 minutes, cycle number 35; in the third stage, the temperature is 72 ℃ and the cycle number is 1 for 10 minutes.
The PCR amplified product is subjected to sequence detection by adopting a second detection primer (the sequence is the same as that of the primer R2), and the pertussis toxin S1 subunit deleted recombinant strain of the pertussis bacillus is obtained after comparison and confirmation and is named 18123.
Example 3
In this example, mRNA sequences, growth curves and PT protein expression amounts of recombinant strains FE3, FE16 and 18123 of bordetella pertussis prepared in example 1 and example 2, and in vitro toxicity of PT proteins of the recombinant strains FE3 and FE16 were studied.
3.1 verification of mRNA sequence of recombinant strains of Bordetella pertussis
Recombinant strains FE3, FE16 and 18123 were cultured by amplification in SSM medium containing 25. Mu.g/mL Kana, and a suitable amount of bacterial liquid was collected and total RNA was extracted using TaKaRa MiniBEST Universal RNA Extraction Kit kit (purchased from TaKaRa Co., ltd.: 9767). PrimeScript was used with primers F2 and R2 as the upstream and downstream primers, respectively, or with primers F3 and R2 as the upstream and downstream primers, respectively TM II High Fidelity One Step RT-PCR Kit (purchased from TaKaRa, cat# R026A) the total RNA extracted was subjected to RNA region-selective reverse transcription amplification comprising the PT-S1 subunit. The PCR amplified product is subjected to sequence detection and comparison by using a first detection primer or a second detection primer, and it is confirmed that R9K/E129G double-site mutation occurs in the translation template of the PT-S1 subunit protein of the recombinant strain FE3, R9K single-site mutation occurs in the translation template of the PT-S1 subunit protein of the recombinant strain FE16, and the translation template of the PT-S1 subunit protein of the recombinant strain 18123 is deleted.
3.2 growth curves of recombinant strains of bordetella pertussis
Recombinant strains 18123, FE3, FE16 and Wild Type (WT) bacteria were inoculated in 250mL shake flasks, respectivelyThe final concentration of the inoculum was controlled to 2 hundred million CFU/mL in SSM medium ranging from the strain (pertussis CS strain) to 50 mL. Placing the shake flask on a constant temperature shake table, continuously culturing at 35deg.C and 220rpm, and collecting bacterial sample at 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 60h, and 72h to detect bacterial liquid OD 600nm The values were plotted and the results are shown in FIG. 2. As can be seen from FIG. 2, compared with the wild type pertussis CS strain, the growth rate of the recombinant strain 18123 is similar, the growth rates of the recombinant strains FE3 and FE16 are slightly lower, and the maximum bacterial concentration of the recombinant strains FE3 and FE16 is higher than that of the wild type strain.
3.3 detection of recombinant strain PT protein ELISA of bordetella pertussis
Recombinant strains 18123, FE3 and FE16 to 50mL SSM medium were inoculated in 250mL shake flasks, respectively, with a final inoculum concentration of 2 hundred million CFU/mL. Culturing continuously on a constant temperature shaking table at 35 ℃ and 220rpm, taking fungus samples at 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 72h, 96h, 120h and 144h respectively, centrifuging at 8000g at 4 ℃ for 10 minutes, and collecting culture supernatants. The samples are respectively diluted by 25 times, 50 times, 100 times, 200 times, 400 times and 800 times, the samples are detected by adopting a PT multi-antibody double-sandwich ELISA method, standard curves of PT protein detection ranges of 1.25-20 ng/mL are drawn by diluting the standard products JNIH-5 times of the pertussis toxin standard products of WHO, the corresponding numerical values of the samples in the ranges are multiplied by dilution times respectively to calculate the average number, the PT content in culture supernatants collected every day is obtained, and therefore, an aging curve is drawn, and the result is shown in figure 3. As can be seen from FIG. 3, the recombinant strains of Bordetella pertussis FE3 and FE16 showed an increase in the secreted PT protein content with increasing culture time, with maximum values of about 800ng/mL and 200ng/mL, respectively. Recombinant strain 18123 detection values were consistently below the lower detection limit.
3.4 in vitro toxicity detection of recombinant strain PT protein of Bordetella pertussis
Recombinant strains FE3, FE16 were evaluated for in vitro toxicity of PT proteins using CHO cell agglutination experiments.
The supernatant cultured to 48 hours in step 3.3 was concentrated 10 times with a 10KD ultrafiltration tube (Merck Co., UFC 9010), sterilized with a 0.22 μm filter as an experimental group, and subjected to blow-molding with a WHO 1 st generation pertussis toxin standard JNIH-5 diluted to one active unit as a control group, 240. Mu.L of each was added to column 1 of a blank 96-well dilution plate, 120. Mu.L of CHO cell culture medium was added to the remaining column as a multiple dilution, 120. Mu.L of each was sequentially added to the next from column 1 to column 12, and the mixture was blow-molded to complete multiple dilution. mu.L per well was then added from the 96-well dilution plate to a 96-well cell plate plated with 3000 CHO cells per well one day in advance, and culture was continued for 48 hours. After the completion of the incubation, the supernatant was discarded from the 96-well cell plate, and the plate was stained with 0.1% crystal violet at room temperature for 10 minutes, and the purified water was gently washed to remove excess dye solution. Observing the agglutination condition of the CHO cells of each group by microscopic examination, wherein the microscopic examination results are shown in graphs A-C in FIG. 4; FIG. 4A shows the agglutination of CHO cells at a PT level of 20 ng/mL; panel B is a microscopic image of CHO cells at 50% agglutination; panel C shows growth of CHO cells in the blank without PT. FIG. 4D shows the statistical results of the minimum PT levels required for CHO cell agglutination in the experimental group (recombinant strains FE3 and FE 16) and the control group (standard JNIH-5), and it can be seen from the figure that the minimum concentration of CHO cell agglutination caused by standard JNIH-5 is 13pg/mL, and the recombinant strains FE3 and FE16 are 795.380ng/mL and 160.950ng/mL, respectively. Therefore, compared with the WHO 1 st generation pertussis toxin standard JNIH-5, the in vitro toxicity of PT proteins expressed by recombinant strains FE3 and FE16 is reduced to 0.0016% and 0.0081% respectively.
3.5 immunoblot analysis of recombinant strain PT protein of bordetella pertussis
The supernatant stock cultured in step 3.3 to 48 hours was mixed with 5 Xprotein loading buffer (purchased from Shanghai Biotechnology Co., ltd., cat# C508320), boiled at 100℃for 10 minutes, subjected to SDS-PAGE protein electrophoresis, and after transfer, blocked overnight with 5% nonfat milk powder at 4℃and incubated with HRP-labeled S1 subunit monoclonal antibody 1B7 at room temperature for 2 hours. The membrane was washed three times with 1 XPBST for 5 minutes, after which the PT-S1 subunit band was detected in a chromogenic instrument. FIG. 5 shows the immunoblotting results of the 1B7 monoclonal antibodies against the PT proteins of recombinant strains 18123, FE3 and FE16, wherein the detection lanes of recombinant strains FE3 and FE16 show bands at the target site of PT-S1 subunit (26 kD), and no relevant bands are detected in the lanes of recombinant strain 18123; the recombinant strain FE3 and FE16 has unchanged epitope and immunogenicity.
The foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Various modifications and alterations of this invention will occur to those skilled in the art. Any and all such simple and equivalent variations and modifications are intended to be included within the scope of this invention.
Claims (10)
1. An attenuated recombinant strain of bordetella pertussis, wherein the recombinant strain of bordetella pertussis is obtained by subjecting a mature S1 subunit of pertussis toxin of a wild-type strain of bordetella pertussis CS to one or more amino acid mutations or a full length deletion of the S1 subunit; the recombinant bordetella pertussis strain has kanamycin resistance; the amino acid sequence of the mature S1 subunit of pertussis toxin of the wild type pertussis bacillus CS strain is shown as SEQ ID No. 1.
2. The recombinant strain of bordetella pertussis according to claim 1, wherein the mature S1 subunit of the pertussis toxin expressed by the recombinant strain comprises the double site mutations R9K and E129G in the amino acid sequence shown in SEQ ID No. 1.
3. The recombinant bordetella pertussis strain according to claim 2, wherein the amino acid sequence of the mature S1 subunit of the pertussis toxin expressed by the recombinant bordetella pertussis strain is shown in SEQ ID No. 2.
4. A recombinant bordetella pertussis strain according to claim 3, wherein the genome of the recombinant bordetella pertussis strain comprises the nucleotide sequence shown in SEQ ID No.3 or comprises a nucleotide sequence having more than 95% homology with the amino acid sequence shown in SEQ ID No.2 after translation.
5. The method for constructing a recombinant strain of bordetella pertussis as defined in claims 1 to 4, comprising the steps of: constructing a recombinant plasmid pB-Kana-gS1 containing PT-S1 subunit mutant genes, and preparing competent cells of wild type pertussis CS strain; preparing the recombinant plasmid pB-Kana-gS1 into a linearized DNA recombinant fragment containing a PT-S1 subunit mutant gene, electrically transferring the linearized DNA recombinant fragment containing the PT-S1 subunit mutant gene into the CS strain competent cells, resuscitating, and screening by kanamycin resistance to obtain a positive strain which is the recombinant strain of the pertussis bacillus with the PT-S1 subunit gene mutation;
or preparing competent cells of wild pertussis CS strain, electrotransferring the pKD46 plasmid into the competent cells of the CS strain to obtain the pertussis CS strain containing the pKD46 plasmid, and preparing the competent cells containing the pKD46 plasmid; constructing a recombinant plasmid pB-delta S1 with deletion of PT-S1 subunit gene; preparing the recombinant plasmid pB-delta S1 into a linearization DNA recombinant fragment without PT-S1 subunit gene, electrically transferring the linearization DNA recombinant fragment without PT-S1 subunit gene into competent cells containing the pKD46 plasmid, resuscitating, and screening by kanamycin resistance to obtain a positive strain which is the recombinant strain of the pertussis bacillus with the PT-S1 subunit gene deleted.
6. The construction method according to claim 5, wherein the construction method of the recombinant plasmid pB-Kana-gS1 comprises the steps of: the method comprises the steps of taking a pBluescript II SK (+) plasmid as a vector skeleton, taking restriction enzyme cutting sites XhoI and BamHI as target fragment connecting sites, and integrating a 5 'homology arm of a PT-S1 subunit gene, a NOS promoter, a Kana resistance gene, a PT-S1 subunit mutant gene and a 3' homology arm of the PT-S1 subunit gene into the pBluescript II SK (+) plasmid through gene synthesis; or (b)
The construction method of the recombinant plasmid pB-delta S1 comprises the following steps: the vector skeleton is pBluescript II SK (+) plasmid, the restriction enzyme sites XhoI and BamHI are used as target fragment connecting sites, and the 5 'homology arm of PT-S1 subunit gene, the Amp promoter, the Kana resistance gene and the 3' homology arm of PT-S1 subunit gene are integrated into the pBluescriptII SK (+) plasmid through gene synthesis.
7. The method of constructing according to claim 5, wherein the method of preparing competent cells of wild-type bordetella pertussis comprises the steps of: inoculating wild pertussis to Stainer-Scholte liquid culture medium, and culturing to OD 600nm After bacterial cells are collected and washed with sterile double distilled water precooled to 2-8 ℃, the bacterial cells are washed 2 times with 10% glycerol precooled to 2-8 ℃, and then sub-packaged with 100 mu L of each tube and stored at-80 ℃.
8. The method of claim 5, wherein the conditions for the electrotransformation are: the electrode spacing of the electric shock cup is 1mm, the capacitance is 25 mu F, the resistance is 200 omega, and the voltage is 2400V.
9. The method of construction according to claim 5, wherein the step of screening for kanamycin resistance comprises: the resuscitated bacterial solution was spread on a 15% sheep blood Borset-Gengou resistant plate containing kanamycin and incubated at 35.+ -. 2 ℃ for 3-5 days.
10. Use of the recombinant strain of bordetella pertussis according to any one of claims 1 to 4 for the preparation of a product for the treatment or/and detection or/and prevention of a pertussis disease.
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