WO2017045281A1 - 一种弧菌通用的基因敲除自杀载体及其应用 - Google Patents

一种弧菌通用的基因敲除自杀载体及其应用 Download PDF

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WO2017045281A1
WO2017045281A1 PCT/CN2015/097202 CN2015097202W WO2017045281A1 WO 2017045281 A1 WO2017045281 A1 WO 2017045281A1 CN 2015097202 W CN2015097202 W CN 2015097202W WO 2017045281 A1 WO2017045281 A1 WO 2017045281A1
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gene
plp12t
vibrio
suicide vector
plp12
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罗鹏
何香燕
刘秋婷
胡超群
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中国科学院南海海洋研究所
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    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • C12N15/03—Bacteria
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/63—Vibrio

Definitions

  • the invention belongs to the field of microbial genetic operations, and particularly relates to a gene knockout suicide vector commonly used by Vibrio and application thereof.
  • Vibrio is the most common heterotrophic bacteria in the marine environment. At least 12 kinds of Vibrio can cause human disease. Among them, Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus are the most serious harm to human health. In addition, many types of Vibrio are also the pathogens of aquatic economic animals, and the vibrio caused by them causes a large economic loss in aquaculture. Although some key pathogenic genes of Vibrio cholerae and Vibrio parahaemolyticus have been discovered very early, scientists still have a serious lag behind the pathogenic bacteria of Enterobacteriaceae. The lack of mature and convenient genetic manipulation technology is the main obstacle to the study of the pathogenic mechanism of Vibrio.
  • the suicide vector is the core of the gene knockout technology based on the bacterial recBCD homologous recombination system.
  • the gene knockout technique that can be successfully used for homologous recombination of bacteria such as Escherichia coli has some obvious defects in Vibrio gene knockout, including: (1) lack of versatility, limited to gene deletion in a certain Vibrio; (2) sacB is widely used as a lethal gene in suicide vectors, but the lethal effect caused by sacB is very weak in Vibrio, resulting in a very low proportion of correct deletion mutants in reverse selection plates, resulting in a large number of deletion mutations.
  • the object of the present invention is to provide a gene knockout suicide vector pLP12T which is common to Vibrio, in view of the deficiencies of the prior art.
  • the gene knockout suicide vector pLP12 of the present invention is a circular vector, which comprises a P BAD promoter, a repressor gene araC, an RP4 transfer initiation site (oriT RP4 ), and a chloramphenicol resistance gene (cat).
  • R6K replication initiation site oriV R6K ⁇
  • MCS multiple cloning site region
  • lethal gene vmi480 said multiple cloning site region (MCS) comprising at least two AhdI cleavage sites, said lethal the nucleotide sequence of the gene of vmi480 bit of 21-617 base sequence shown in SEQ ID NO.1, said vmi480 lethal gene located downstream of the P BAD promoter and is under the control of P BAD promoter.
  • the gene knockdown suicide vector pLP12 has a nucleotide sequence as shown in SEQ ID NO. 1, and contains a 3871 bp base.
  • the present invention also provides a gene knockout suicide vector pLP12T, which is a linear vector, which is characterized in that the above gene knockout suicide vector pLP12 is digested with two AhdI restriction sites in the multiple cloning site region by AhdI.
  • the resulting linear vector is the gene knockout suicide vector pLP12T.
  • the gene knockdown suicide vector pLP12T has a nucleotide sequence as shown in SEQ ID NO. 2 and contains a 3848 bp base.
  • the P BAD promoter system (including the P BAD promoter and the repressor gene araC) is a tightly controllable expression promoter system, and glucose can completely block the expression of the downstream lethal gene vmi480, and the expression of vmi480 under the induction of arabinose Activated.
  • the expression level of vmi480 can be regulated depending on the concentration of arabinose induction.
  • oriT RP4 is an AT-rich sequence, and the suicide plasmid forms a single-stranded transfer from this site under the action of the donor bacteria providing a transfer mechanism.
  • the chloramphenicol resistance gene provides a resistance marker for the first integrated plasmid screen.
  • oriV R6K ⁇ is the starting site for self-replication of the plasmid.
  • vmi480 Under the control of the P BAD promoter system, vmi480 can express or inhibit expression. When subjected to reverse screening, vmi480 is activated to express Vmi480 under the induction of arabinose, which is highly toxic. Only the cells that lost the plasmid after the second homologous recombination can survive. The result of the second recombination is that the original cell divides to produce a wild-type cell and a gene-deficient cell, thereby achieving gene knockout.
  • the multiple cloning site region facilitates digestion of the plasmid to produce a suicide vector that can be ligated to the PCR product that is also digested. MCS contains two AhdI restriction sites, which can produce linear suicide T vectors when digested with AhdI. In this case, the PCR product is not required to be digested, and the vector is directly ligated after purification.
  • the invention also provides the use of the gene knockout suicide vector pLP12T in gene knockout.
  • the gene knockout suicide vector pLP12T is used in Vibrio gene knockout.
  • the in-frame deletion fusion fragment of the gene of interest is ligated to the gene knockout suicide vector pLP12T to obtain pLP12T carrying the in-frame deletion fusion fragment of the gene of interest, designated as pLP12T-X, and pLP12T-X is transformed into the donor bacteria. Then, the donor bacteria and the recipient bacteria are mixed, and pLP12T-X is transferred into the recipient bacteria by zygosin. After selection by chloramphenicol, only cells in which pLP12T-X is integrated into the target site of the recipient bacterium can survive.
  • the target gene insertion mutant strain of pLP12T-X integrated into the target site of the recipient genomic genome and then inserting the mutant strain of this target gene under the reverse selection pressure of arabinose-induced vmi480 gene production toxin Vmi480, only occurs for the second time
  • the homologous recombination loses the cells of the suicide vector part to survive.
  • the original gene insertion mutant strain can produce a gene-deficient cell and a normal wild-type cell, so the clones grown on the reverse selection plate are mainly For the target gene deletion clone and the normal wild type cell clone, after screening, the target gene can be screened by PCR detection. The clone is deleted, and finally the strain for knocking off the target gene is obtained, and the strain lacking the target gene is obtained.
  • the recipient strain is Vibrio.
  • the donor bacteria is defective Escherichia coli ⁇ 2163.
  • the use of defective Escherichia coli ⁇ 2163 as a donor strain does not require consideration of whether the recipient bacteria have antibiotic resistance, thus greatly facilitating the application of the junction-based gene knockout method.
  • the present invention Compared with existing suicide vectors and methods of use, the present invention has the following beneficial effects:
  • Vmi480 A novel reverse selection gene vmi480 was used in the knockout suicide vector of the present invention to replace the commonly used sacB gene.
  • Vmi480 has the advantages of strong lethal effect, extensive lethality, and lethal effects are not affected by NaCl. This lays the foundation for the gene knockout suicide vector of the present invention widely used for gene knockout of Vibrio halophilicus.
  • the two cloning sites of the knockout suicide vector of the present invention introduce two AhdI sites, which can form a T vector after digestion, and are used repeatedly after forming a vector.
  • the PCR product does not need to be digested, and does not need to consider the enzyme cleavage site, which greatly saves time.
  • the knockout suicide vector of the present invention eliminates a large number of redundant portions in the general suicide plasmid, and the suicide vector becomes small, which is advantageous for increasing the frequency of the junction transfer and the probability of correct integration.
  • Figure 1 shows the physical map of the gene knockout suicide vector pLP12, which is commonly used by Vibrio. When the enzyme is cut with AhdI, the suicide vector pLP12T can be formed.
  • Figure 2 is a flow chart showing the construction of a gene knockout suicide vector pLP12T which is common to Vibrio.
  • Figure 3 shows the detection of a total of 6 gene knockout results of four different Vibrio species using the Vibrio universal gene knockout suicide vector.
  • M molecular weight Marker
  • 1-3 detection results corresponding to the vah gene wild strain, insertion mutant strain, and deletion mutant strain of Vibrio alginolyticus E0601
  • 4-6 wild strain of degS gene corresponding to Vibrio cholerae HN375, respectively Detection results of insertion mutants and deletion mutants
  • 7-9 detection results corresponding to vasC gene wild strain, insertion mutant strain, and deletion mutant strain of Vibrio cholerae HN375
  • 10-12 corresponding to the accessory hemolysis arc
  • 13-15 the detection results of the ascS gene wild strain, the insertion mutant strain, and the deletion mutant strain corresponding to Vibrio parahaemolyticus E06135
  • 16- 18 Test results corresponding to
  • Example 1 is a construction of a gene knockout suicide vector pLP12T which is common to Vibrio
  • Examples 2-7 are applications of a suicide vector pLP12T in a representative Vibrio gene knockout.
  • Vibrio alginolyticus is widely distributed in marine and estuarine environments. It is the most common species of Vibrio isolated, and it is also a conditional pathogen of many aquaculture animals. The outbreak of vibriosis can cause huge economic losses.
  • Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus are the most serious types of Vibrio species that are harmful to human health.
  • these four Vibrio species also have large genetic differences between each other. Therefore, these four Vibrio species were selected as typical representatives to verify the versatility of Vibrio gene knockout suicide vector pLP12T in Vibrio gene knockout.
  • PCR amplification using the primer pSW23T-F/pSW23T-R, plasmid pSW23T (provided by Vincent Burrus, source document Demarre G, et al. Research in Microbiology, 2005, 156: 245-255) as a template It is a high-fidelity DNA polymerase PrimeSTAR of TaKaRa (unless specifically stated, the polymerase used in the present invention is this enzyme).
  • the PCR amplification conditions are referred to the enzyme instructions.
  • a PCR product was obtained which contained a fragment of oriV R6K ⁇ , oriT RP4 , cat, MCS sites.
  • PCR amplification was carried out using the primer pSW25T-F/pSW25T-R using pSW25T-ccdB (provided by Vincent Burrus, source http://openwetware.org/wiki/Mazel/PSW25T-ccdB.docx) as a template.
  • a PCR product was obtained which contained the P TAC promoter, ccdB and a fragment comprising an AhdI restriction site.
  • Plasmid pLP10 T4 DNA ligase
  • Escherichia coli DH5 ⁇ pir cells LB plates (containing 20 ⁇ g/ml chloramphenicol) were applied.
  • the clones on the plates were screened by primer pair pLP10L-TF1/pLP10L-TR1 and pLP10L-TF2/pLP10L-TR2, and the PCR products of correct positive clones were 1065 bp and 537 bp, respectively.
  • the PCR fragment was further sequenced to confirm the correct clone, which contained the ligation product-plasmid pLP10.
  • PCR was carried out using the genome of Escherichia coli LP79 (construction method reference inventor's article, Carraro N, Matteau D, Luo P, et al. PLOS Genetics, 2014, 10(10): e1004714) using vmi480-F/vmi480-R Amplification, a complete fragment of the vmi480 gene was obtained, and the nucleotide sequence thereof is shown in nucleotide sequences 21 to 617 of SEQ ID NO. Escherichia coli LP79 was previously obtained by conjugative transfer to obtain the gene island MGIVmi1 from Vibrio mimicus VM573, which contains the vmi480 lethal gene.
  • PCR fragment pLP10-1 and vmi480 gene fragment were digested and purified by NdeI/XhoI, respectively, and then ligated with T4 DNA ligase.
  • the ligation product (plasmid pLP11) was transformed into E. coli DH5 ⁇ pir cells, and coated with LB plate (including 20 ⁇ g/ml chloramphenicol, 0.3% D-glucose). The clones on the plate were screened by PCR with pLP11L-TF/pLP11L-TR primers.
  • the positive PCR product was cloned 515 bp in length, and the PCR fragment was sequenced to confirm the correct clone.
  • the correct clone contained the ligation product-plasmid pLP11. Expand the correct clone and culture the plasmid pLP11.
  • pLP11-1 and pBAD30-1 were recombined in vitro using the seamless cloning kit (Vazyme), and the recombinant product (plasmid pLP12) was transformed into E. coli DH5 ⁇ pir cells and coated with LB plates (containing 20 ⁇ g/ml chloramphenicol, 0.3% D). -glucose).
  • the clones on the plate were screened by pLP12L-TF/pLP12L-TR primers.
  • the positive clones were 1104 bp in length, and the PCR fragments were sequenced to confirm the correct clone.
  • the correct clone contained the recombinant product-plasmid pLP12.
  • the correct clone was expanded and the plasmid pLP12 was extracted, which is a gene knockout suicide vector pLP12 which is common to Vibrio, and its nucleotide sequence is shown in SEQ ID NO. The specific structure is shown in Figure 1.
  • the gene knockout suicide vector pLP12 was used as a template, and PCR amplification was carried out to amplify the primer STVU-F/STVU-R.
  • the amplified product was purified by PCR product purification kit and then digested with AhdI. After digestion, it was purified by DNA concentration kit (Zhong Ding) to obtain a linearized Vibrio gene knockout suicide vector pLP12T (its nucleotide The sequence is as shown in SEQ ID NO. 2).
  • the 3' end of the pLP12T double strand has a prominent base T, and thus can be directly ligated to the PCR product produced by amplification of a common DNA polymerase. Neither need to be digested.
  • the second round of PCR uses primers vah-MF1/vah-MR2, using ordinary Taq DNA polymerase (TaKaRa), PCR amplification conditions, PCR amplification conditions, and reference enzyme specifications, thereby obtaining a second round of PCR products.
  • TaKaRa Taq DNA polymerase
  • E. coli ⁇ 2163 (pLP12-vah) and the recipient strain Vibrio alginolyticus E0601 were cultured overnight.
  • 100 ⁇ L of donor bacteria was mixed with 100 ⁇ L of recipient bacteria, centrifuged at 8000 g for 2 min, the supernatant was discarded, 400 ⁇ L of fresh LB medium was added and resuspended, and centrifuged again at 8000 g for 2 min. The supernatant was carefully discarded.
  • the suspension was resuspended by adding 10 ⁇ L of fresh LB medium, and the entire suspension was inhaled and dropped on an LB agar plate. After the ultra-clean typhoon was dried, incubate at 37 ° C for 6 hr.
  • the culture was thoroughly suspended in 1 ml of fresh LB medium, and 100 ⁇ L of a coated LB agar plate (containing 0.3% D-glucose, 10 ⁇ g/ml chloramphenicol) was taken and cultured at 37 ° C overnight. On this plate, only the first homologous recombination occurred, and the inserted mutant Vibrio alginolyticus cells with the chromosomal integration of the suicide plasmid pLP12-vah could grow. Randomly picked clones for PCR identification, PCR primers vah-MF1/vah-MR2, and correct clones produced two bands of 1218 bp and 660 bp (lane 2 in Figure 3) to obtain a vah insertion mutant.
  • vah insertion mutant was cultured in LB (0.3% D-glucose) for 3 hours, and then diluted by a gradient, 100 ⁇ L of each of LB agar plates (0.2% L-arabinose) was applied, and cultured at 37 ° C overnight. On this plate, only the second homologous weight occurs Group, chromosome loss integrated cell suicide plasmid pLP12-vah cells can survive. After the second recombination of the bacterial cells, a cell with a deletion of the vah gene and a cell containing the wild-type vah gene are produced.
  • An in-frame deletion fusion fragment of the degS gene of Vibrio cholerae HN375 was obtained by overlapping PCR amplification.
  • the first round of PCR primer pairs were degs-MF1/degs-MR1, degs-MF2/degs-MR2, and the template was Vibrio cholerae HN375 genomic DNA.
  • the two PCR products were mixed in equal volumes and purified as a template for the second round of PCR.
  • the second round of PCR uses the primer degs-MF1/degs-MR2, which uses a common Taq DNA polymerase to obtain a second round of PCR product.
  • the second round of PCR product described above was directly ligated to the suicide vector pLP12T.
  • the ligation product (plasmid pLP12-degS) was transformed into E. coli DH5 ⁇ pir cells and coated with LB plates (containing 20 ⁇ g/ml chloramphenicol, 0.3% D-glucose).
  • the clones on the plates were screened by degs-MF1/degs-MR2 primers, and the correct clones produced a 926 bp amplified band.
  • the PCR fragment was further sequenced to confirm the correct clone, and the correct clone contained the ligation product-plasmid pLP12-degS.
  • the correct clone was expanded and the plasmid pLP12-degS was extracted.
  • pLP12-degS was electrotransformed into E. coli ⁇ 2163 cells.
  • the electroporation conditions and the cell culture conditions after the electric shock were the same as in Example 2. Any clone was picked, purified and stored to obtain a donor strain containing the suicide plasmid pLP12-degS - Escherichia coli ⁇ 2163 (pLP12-degS).
  • E. coli ⁇ 2163 (pLP12-degS) and the recipient strain Vibrio cholerae HN375 were cultured overnight.
  • 100 ⁇ L of donor bacteria was mixed with 100 ⁇ L of recipient bacteria, centrifuged at 8000 g for 3 min, the supernatant was discarded, 500 ⁇ L of fresh LB medium was added and resuspended, and centrifuged again at 8000 g for 2 min. The supernatant was carefully discarded.
  • the suspension was resuspended by adding 10 ⁇ L of fresh LB medium, and the entire suspension was inhaled and dropped on an LB agar plate. After the ultra-clean typhoon was dried, incubate at 37 ° C for 7 hr.
  • the degS insertion mutant was cultured in LB (0.3% D-glucose) for 3 hours, and then diluted by a gradient, 100 ⁇ L of each of LB agar plates (0.2% L-arabinose) was applied, and cultured at 37 ° C overnight. On this plate, only the second homologous recombination occurred, and the cells of the chromosome-losing integrated state suicide plasmid pLP12-degS were able to survive. After the second recombination of the bacterial cells, a cell with a deletion of the degS gene and a cell containing the wild-type degS gene are produced.
  • PCR was performed on LB agar plates (0.2% L-arabinose) by degS-MF1/degS-MR2 primers, and the correct degS gene deletion clone produced a 926 bp band (lane 6 in Figure 3).
  • the wild-type clone formed by the second recombination produced a 1496 bp band (lane 4 in Figure 3).
  • the correct degS gene deletion clone was verified by PCR product sequencing, and the degS gene deletion strain was obtained, thereby completing the degS gene knockout of Vibrio cholerae. In this example, 8 of the 16 clones randomly picked on the reverse selection plate ( 10-1 dilution) were the correct degS gene deletion clones.
  • pLP12-vasC was electrotransformed into E. coli ⁇ 2163 cells.
  • the electroporation conditions and the cell culture conditions after the electric shock were the same as in Example 2. Any clone was picked, purified and stored to obtain a donor strain containing the suicide plasmid pLP12-vasC - Escherichia coli ⁇ 2163 (pLP12-vasC).
  • E. coli ⁇ 2163 (pLP12-vasC) and the recipient strain Vibrio cholerae HN375 were cultured overnight.
  • 100 ⁇ L of donor bacteria was mixed with 100 ⁇ L of recipient bacteria, centrifuged at 8000 g for 2 min, the supernatant was discarded, 600 ⁇ L of fresh LB medium was added to resuspend, centrifuged again at 8000 g for 2 min, and the supernatant was carefully discarded.
  • the suspension was resuspended by adding 10 ⁇ L of fresh LB medium, and the entire suspension was inhaled and dropped on an LB agar plate. After the ultra-clean typhoon was dried, incubate at 37 ° C for 8 hr.
  • the culture was thoroughly suspended in 1 ml of fresh LB medium, and 100 ⁇ L of a coated LB agar plate (containing 0.3% D-glucose, 10 ⁇ g/ml chloramphenicol) was taken and cultured at 37 ° C overnight. On this plate, only the first homologous recombination occurred, dyeing The chromosomal integration of the insertional mutant Vibrio cholerae cells with the suicide plasmid pLP12-vasC can be grown. The clones were randomly picked for PCR identification, PCR primer vasC-MF1/vasC-MR2, and the correct clones produced two bands of 1630 bp and 925 bp (lane 8 in Figure 3) to obtain a vasC insertion mutant.
  • vasC insertion mutant was cultured in LB (0.3% D-glucose) for 3 hours, and then diluted by a gradient, 100 ⁇ L of each of LB agar plates (0.2% L-arabinose) was applied, and cultured at 37 ° C overnight. On this plate, only the second homologous recombination occurred, and the cells of the chromosome-integrated suicide plasmid pLP12-vasC were able to survive. The bacterial cell is recombined for a second time to produce a vasC gene-deficient cell and a cell containing the wild-type vasC gene.
  • PCR was performed on LB agar plates (0.2% L-arabinose) by vasC-MF1/vasC-MR2 primers, and the correct vasC gene deletion clone produced a 925 bp band (lane 9 in Figure 3).
  • the wild-type clone formed by the second recombination produced a 1630 bp band (lane 7 in Figure 3).
  • the correct vasC gene deletion clone was verified by PCR product sequencing, and the vasC gene deletion strain was obtained, thereby completing the vasculin gene knockout of Vibrio cholerae.
  • 7 of the 16 clones randomly picked on the reverse selection plate ( 10-1 dilution) were the correct vasC gene deletion clones.
  • An in-frame deletion fusion fragment of the pilO gene of Vibrio parahaemolyticus E0680 was obtained by overlapping PCR amplification.
  • the first round of PCR primer pairs were pilO-MF1/pilO-MR1, pilO-MF2/pilO-MR2, and the template was Vibrio parahaemolyticus E0680 genomic DNA.
  • an equal volume was mixed as a second round of PCR template.
  • the second round of PCR primers pilO-MF1/pilO-MR2 was performed using ordinary Taq DNA polymerase. A second round of PCR product was obtained.
  • pLP12-pilO was electrotransformed into E. coli ⁇ 2163 cells.
  • the electroporation conditions and the cell culture conditions after the electric shock were the same as in Example 2. Any clone was picked, purified and stored to obtain a donor strain containing the suicide plasmid pLP12-pilO-E. coli ⁇ 2163 (pLP12-pilO).
  • E. coli ⁇ 2163 (pLP12-pilO) and the recipient strain Vibrio parahaemolyticus E0680 were cultured overnight.
  • 100 ⁇ L of donor bacteria was mixed with 100 ⁇ L of recipient bacteria, centrifuged at 8000 g for 2 min, the supernatant was discarded, 400 ⁇ L of fresh LB medium was added to resuspend, centrifuged again at 8000 g for 2 min, and the supernatant was carefully discarded. Resuspend in 10 ⁇ L of fresh LB medium and absorb all the resuspension Into the LB agar plate. After the ultra-clean typhoon was dried, incubate at 37 ° C for 8 hr.
  • the culture was thoroughly suspended in 1 ml of fresh LB medium, and 100 ⁇ L of a coated LB agar plate (containing 0.3% D-glucose, 10 ⁇ g/ml chloramphenicol) was taken and cultured at 37 ° C overnight. On this plate, only the first homologous recombination occurred, and the mutated integration of the suicide plasmid pLP12-pilO into the mutant Vibrio parahaemolyticus cells can grow.
  • the clones were randomly picked for PCR identification, PCR primers pilO-MF1/pilO-MR2, and the correct clones produced two bands of 1214 bp and 815 bp (lane 11 in Figure 3) to obtain pilO insertion mutants.
  • the pilO insertion mutant was cultured in LB (0.3% D-glucose) for 3 hours, and then diluted in a gradient, 100 ⁇ L of each of LB agar plates (0.2% L-arabinose) was applied, and cultured at 37 ° C overnight. On this plate, only the second homologous recombination occurred, and the cell with the chromosome loss integrated state suicide plasmid pLP12-pilO survived. A second recombination of bacterial cells produces a pilO-deficient cell and a cell containing the wild-type pilO gene.
  • the ascS gene of Vibrio parahaemolyticus E06135 was amplified by overlapping PCR to obtain an ascS in-frame deletion fusion fragment.
  • the first round of PCR primer pairs were ascS-MF1/ascS-MR1, ascS-MF2/ascS-MR2, and the template was Vibrio parahaemolyticus E06135 genomic DNA.
  • the two PCR products were mixed in equal volumes and purified for use as a template for the second round of PCR.
  • the ligation product (plasmid pLP12-ascS) was transformed into E. coli DH5 ⁇ pir cells and coated with LB plates (containing 20 ⁇ g/ml chloramphenicol, 0.3% D-glucose). The clones on the plates were screened by the ascS-MF1/ascS-MR2 primers, and the correct clones produced a 703 bp amplified band. The PCR fragments were then sequence verified to determine the correct clone, which contained the ligation product-plasmid pLP12-ascS. Expand the correct clone and culture the plasmid pLP12-ascS.
  • pLP12-ascS was electroporated into E. coli ⁇ 2163 cells.
  • the electroporation conditions and the cell culture conditions after the electric shock were the same as in Example 2. Any clone was picked, purified and stored to obtain a donor strain containing the suicide plasmid pLP12-ascS, Escherichia coli ⁇ 2163 (pLP12-ascS).
  • E. coli ⁇ 2163 (pLP12-ascS) and the recipient strain Vibrio parahaemolyticus E06135 were cultured overnight.
  • 100 ⁇ L of donor bacteria was mixed with 100 ⁇ L of recipient bacteria, centrifuged at 8000 g for 2 min, the supernatant was discarded, 400 ⁇ L of fresh LB medium was added and resuspended, and centrifuged again at 8000 g for 2 min. The supernatant was carefully discarded.
  • the suspension was resuspended by adding 10 ⁇ L of fresh LB medium, and the entire suspension was inhaled and dropped on an LB agar plate. After the ultra-clean typhoon was dried, incubate at 37 ° C for 8 hr.
  • the culture was thoroughly suspended in 1 ml of fresh LB medium, and 100 ⁇ L of a coated LB agar plate (containing 0.3% D-glucose, 10 ⁇ g/ml chloramphenicol) was taken and cultured at 37 ° C overnight. On this plate, only the first homologous recombination occurred, and the inserted mutant Vibrio parahaemolyticus cells with the chromosomal integration of the suicide plasmid pLP12-ascS were able to grow.
  • the ascS insertion mutant was cultured in LB (0.3% D-glucose) for 4 hours, diluted in a gradient, and 100 ⁇ L of each of the coated LB agar plates (0.2% L-arabinose) was taken and cultured overnight at 37 °C. On this plate, only the second homologous recombination occurred, and the cells with the chromosome loss integrated state suicide plasmid pLP12-degS survived. After the second recombination of the bacterial cells, a cell lacking the ascS gene and a cell containing the wild-type ascS gene are produced.
  • pLP12-impB was electrotransformed into E. coli ⁇ 2163 cells.
  • the electroporation conditions and the cell culture conditions after the electric shock were the same as in Example 2. After overnight culture, any clone was picked, purified and stored to obtain a donor strain containing the suicide plasmid pLP12-impB-E. coli ⁇ 2163 (pLP12-impB).
  • E. coli ⁇ 2163 (pLP12-impB) and the recipient strain Vibrio vulnificus ATCC 27562 were cultured overnight.
  • 100 ⁇ L of donor bacteria was mixed with 100 ⁇ L of recipient bacteria, centrifuged at 8000 g for 3 min, the supernatant was discarded, 600 ⁇ L of fresh LB medium was added to resuspend, centrifuged again at 8000 g for 3 min, and the supernatant was carefully discarded.
  • the suspension was resuspended by adding 10 ⁇ L of fresh LB medium, and the entire suspension was inhaled and dropped on an LB agar plate. After the ultra-clean typhoon was dried, incubate at 37 ° C for 7 hr.
  • the culture was thoroughly suspended in 1 ml of fresh LB medium, and 100 ⁇ L of a coated LB agar plate (containing 0.3% D-glucose, 10 ⁇ g/ml chloramphenicol) was taken and cultured at 37 ° C overnight. On this plate, only the inserted mutant Vibrio vulnificus cells with the suicide plasmid pLP12-impB can be grown after the first homologous recombination. The clones were randomly picked for PCR identification, PCR primers impB-MF1/impB-MR2, and the correct clones produced two bands of 1290 bp and 909 bp (lane 17 in Figure 3), and the impB insertion mutant was obtained.
  • the impB insertion mutant was cultured in LB (0.3% D-glucose) for 3 hours, and diluted in a gradient, 100 ⁇ L of each of LB agar plates (0.2% L-arabinose) was applied, and cultured at 37 ° C overnight. On this plate, only the second homologous recombination occurred, and the cells of the chromosome-losing integrated state suicide plasmid pLP12-impB survived. After the second recombination of the bacterial cells, an impB gene-deficient cell and a wild-type impB gene-containing cell are produced.

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Abstract

提供了一种弧菌通用的用于基因敲除的自杀载体、其构建方法及其在弧菌基因敲除中的应用。该自杀载体pLP12为环状载体，包括PBAD启动子、阻遏蛋白基因 araC、RP4转移起始位点、氯霉素抗性基因、R6K复制起始位点、多克隆位点区和致死基因vmi480，其中多克隆位点区至少包含两个AhdI酶切位点。该自杀载体pLP12经AhdI酶切后形成线性化自杀载体pLP12T。

Description

一种弧菌通用的基因敲除自杀载体及其应用 技术领域：
本发明属于微生物遗传操作领域，具体涉及一种弧菌通用的基因敲除自杀载体及其应用。
背景技术：
弧菌是海洋环境中最为常见的异养细菌，至少有12种弧菌可引起人类致病，其中霍乱弧菌、创伤弧菌、副溶血弧菌对人类健康危害最为严重。此外不少种类的弧菌也是水产经济动物病原，其引发的弧菌病导致水产养殖大量经济损失。尽管很早已经发现了霍乱弧菌和副溶血弧菌某些关键致病基因，科学家对弧菌致病机理的认识仍然严重滞后于肠杆菌科的致病菌。缺乏成熟、便捷的遗传操作技术是弧菌分子致病机理研究的主要障碍。
基于细菌recBCD同源重组系统的基因敲除技术是目前在多种细菌中可以实现的技术。在这种技术中，目的基因的框内缺失融合片段首先通过交叠PCR扩增获得，PCR产物与自杀载体采用相同的酶酶切纯化后再连接，并转化大肠杆菌，获得带有外源片段的自杀质粒(载体)，该自杀质粒再经由供体菌接合输入到受体靶细菌中。该质粒由于自身的限制并不能在靶细菌中复制，在正向选择压力下(通常是质粒上的抗性基因)，只有基因组通过同源重组整合了自杀质粒的靶细菌才能存活下来，形成插入突变；在反向选择压力下，插入突变细胞发生第二次同源重组，整合态的自杀质粒脱离染色体，理论上形成一个野生型细胞和一个基因缺失突变细胞，从而实现靶基因的敲除。由此可见，自杀载体是基于细菌recBCD同源重组系统的基因敲除技术的核心。
目前基于细菌recBCD同源重组系统的基因敲除技术在弧菌中的应用还非常少见，仅有一些偶然成功的报道。可以成功用于大肠杆菌等细菌的同源重组的基因敲除技术在弧菌基因敲除中存在一些明显的缺陷，包括：(1)缺乏通用性，仅限于某一个弧菌中的基因缺失；(2)sacB被广泛用作自杀载体的致死基因，但sacB所引发的致死效应在弧菌中十分弱，导致反向选择平板中正确的缺失突变克隆出现的比例非常低，造成大量的缺失突变筛选工作；(3)需要对目的片段交叠PCR产物及自杀质粒进行双酶切，费时费力；(4)自杀质粒本身较大，且带有mobI移动基因，不仅造成质粒接合转移的效率低，而且有可能引起非特异性整合。(5)受体菌必须具有与供体菌及自杀载体不同的抗性标记，限制了常规技术的应用。因此，当用于弧菌基因敲除时，现有自杀载体表现出明显的局限性，迫切需要发展新的通用型自杀载体以克服上述缺陷，实现基于细菌recBCD同源重组系统的基因敲除技术在弧菌中的广泛 应用。
发明内容：
本发明的目的是针对现有技术的不足，提供一种弧菌通用的基因敲除自杀载体pLP12T。
本发明的基因敲除自杀载体pLP12，为环状载体，其特征在于，包括PBAD启动子、阻遏蛋白基因araC、RP4转移起始位点(oriTRP4)、氯霉素抗性基因(cat)、R6K复制起始位点(oriVR6Kγ)、多克隆位点区(MCS)和致死基因vmi480，所述的多克隆位点区(MCS)至少包含两个AhdI酶切位点，所述的致死基因vmi480的核苷酸序列如SEQ ID NO.1的第21-617位碱基序列所示，所述的致死基因vmi480位于PBAD启动子下游并处于PBAD启动子控制之下。
优选，所述的基因敲除自杀载体pLP12，其核苷酸序列如SEQ ID NO.1所示，含有3871bp的碱基。
本发明还提供了一种基因敲除自杀载体pLP12T，为线性载体，其特征在于，是将上述基因敲除自杀载体pLP12用AhdI酶切多克隆位点区中的两个AhdI酶切位点后得到的线性载体，即为基因敲除自杀载体pLP12T。
优选，所述的基因敲除自杀载体pLP12T，其核苷酸序列如SEQ ID NO.2所示，含有3848bp的碱基。
所述的PBAD启动子系统(包括PBAD启动子和阻遏蛋白基因araC)属严密型可控表达启动子系统，葡萄糖可以完全封闭下游致死基因vmi480的表达，在阿拉伯糖的诱导下，vmi480表达被激活。根据阿拉伯糖诱导浓度不同，vmi480的表达量可受调控。oriTRP4为一段富含AT序列，在供体菌提供转移机制的作用下，自杀质粒从此位点开始形成单链转移。氯霉素抗性基因提供第一次整合质粒筛选的抗性标记。oriVR6Kγ为质粒进行自我复制的起始位点。在PBAD启动子系统的控制下，vmi480可以表达或抑制表达。当进行反向筛选时，在阿拉伯糖诱导下vmi480激活表达Vmi480，其具有强烈毒性。只有发生第二次同源重组丢失质粒的细胞才能存活，第二次重组的结果就是原细胞分裂后产生一个野生型细胞和一个基因缺失型细胞，从而实现基因敲除。多克隆位点区(MCS)方便对质粒进行酶切产生可以与同样进行酶切的PCR产物连接的自杀载体。MCS包含二个AhdI酶切位点，当采用AhdI酶切后可产生线性的自杀型T载体。在这种情况下，不需要对PCR产物进行酶切，纯化后就直接连接该载体。
本发明还提供了基因敲除自杀载体pLP12T在基因敲除中的应用。
优选，所述的基因敲除自杀载体pLP12T在弧菌基因敲除中的应用。
进一步优选，将目的基因的框内缺失融合片段与基因敲除自杀载体pLP12T连接，得到携带目的基因的框内缺失融合片段的pLP12T，命名为pLP12T-X，pLP12T-X经转化进入供体菌中，然后将此供体菌与受体菌混合，经接合作用将pLP12T-X转移进入受体菌，经氯霉素选择，只有pLP12T-X整合到受体菌基因组靶位点的细胞才能存活，获得pLP12T-X整合到受体菌基因组靶位点的目的基因插入突变型菌株，然后此目的基因插入突变型菌株在阿拉伯糖诱导vmi480基因产生毒素Vmi480的反向选择压力下，只有发生第二次同源重组丢失了自杀载体部分的细胞才能存活，第二次重组后，原目的基因插入突变型菌株可产生一个基因缺失细胞和一个正常的野生型细胞，因此反向选择平板上生长的克隆主要为目的基因缺失克隆和正常的野生型细胞克隆，经过筛选，如通过PCR检测可以筛选出目的基因缺失的克隆，从而最终达到将目的基因敲除目的，获得目的基因缺失的菌株。
所述的受体菌为弧菌。
所述的供体菌为缺陷型大肠杆菌β2163。采用缺陷型大肠杆菌β2163作为供体菌，不需要考虑受体菌是否具有抗生素抗性，因此大大方便了基于接合的基因敲除方法的应用。
与已有的自杀载体及使用方法相比，本发明具有以下有益效果：
(1)本发明的基因敲除自杀载体中采用了全新的反向选择基因vmi480，用于替代常用的sacB基因。vmi480具有致死作用强、致死对象广泛、致死效应的发挥不受NaCl影响的优点。这为本发明的基因敲除自杀载体广泛用于嗜盐弧菌基因敲除奠定了基础。
(2)本发明的基因敲除自杀载体的多克隆位点引入了两个AhdI位点，酶切后可形成T载体，一次形成载体后，反复使用。PCR产物不用酶切，也不用考虑酶切位点，大大节省了时间。
(3)本发明的基因敲除自杀载体摒除了一般自杀质粒中大量冗余部分，自杀载体变小，有利于提高接合转移的频率以及正确整合的机率。
(4)基于PBAD启动子以及致死基因的高效性，反向选择平板上缺失突变克隆的比率大大提高，大量减少了筛选克隆的工作。
附图说明
图1为弧菌通用的基因敲除自杀载体pLP12物理图谱，当采用AhdI酶切后即可形成自杀载体pLP12T。
图2为弧菌通用的基因敲除自杀载体pLP12T构建流程图。
图3为应用弧菌通用基因敲除自杀载体对4种不同弧菌总计6个基因敲除结果检测。M：分子量Marker；1-3：分别对应于溶藻弧菌E0601的vah基因野生株、插入突变株、缺失突变株的检测结果；4-6：分别对应于霍乱弧菌HN375的degS基因野生株、插入突变株、缺失突变株的检测结果；7-9：分别对应于霍乱弧菌HN375的vasC基因野生株、插入突变株、缺失突变株的检测结果；10-12：分别对应于副溶血弧菌E0680的pilO基因野生株、插入突变株、缺失突变株的检测结果；13-15：分别对应于副溶血弧菌E06135的ascS基因野生株、插入突变株、缺失突变株的检测结果；16-18：分别对应于创伤弧菌ATCC 27562的impB基因野生株、插入突变株、缺失突变株的检测结果：N：代表用水做模板的阴性对照检测结果。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是：在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
以下实施例1为弧菌通用的基因敲除自杀载体pLP12T构建，实施例2-7为自杀载体pLP12T在代表性弧菌基因敲除中的应用。溶藻弧菌广泛分布于海洋及河口环境中，是分离弧菌中最为常见的种类，同时也是多种水产养殖动物的条件致病菌，其导致的弧菌病爆发可引起巨大的经济损失。霍乱弧菌、创伤弧菌、副溶血弧菌是对人类健康危害最为严重弧菌种类。此外，这四种弧菌彼此间也具有较大的遗传差异。因此选用这四种弧菌作为典型代表，用于验证弧菌通用的基因敲除自杀载体pLP12T在弧菌基因敲除中的通用性。
本实施例中所有PCR扩增引物见表1。
表1本发明中所采用的引物
Figure PCTCN2015097202-appb-000001
Figure PCTCN2015097202-appb-000002
Figure PCTCN2015097202-appb-000003
*划线部分为酶切位点
实施例1弧菌通用的基因敲除自杀载体pLP12T的构建(流程如图2所示)
(1)以质粒pSW23T(Vincent Burrus提供，来源文献Demarre G,et al.Research in Microbiology,2005,156:245–255)为模板，采用引物pSW23T-F/pSW23T-R进行PCR扩增，聚合酶为TaKaRa公司高保真DNA聚合酶PrimeSTAR(除非特别说明，本发明中所用聚合酶均为此酶)。PCR扩增条件参考酶说明书。得到PCR产物，该PCR产物包含oriVR6Kγ、oriTRP4、cat、MCS位点的片段。
(2)以pSW25T-ccdB(Vincent Burrus提供，来源http://openwetware.org/wiki/Mazel/PSW25T-ccdB.docx)为模板，采用引物pSW25T-F/pSW25T-R进行PCR扩增。得到PCR产物，该PCR产物包含PTAC启动子、ccdB以及包含一个AhdI酶切位点片段。
(3)分别对上述步骤(1)和(2)的两种PCR产物进行EcoRI/SphI双酶切，酶切产物再采用PCR产物纯化试剂盒(Axygen)进行纯化。
(4)两个纯化产物以T4DNA连接酶(TaKaRa)进行连接，连接产物(质粒pLP10)转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素)。以引物对pLP10L-TF1/pLP10L-TR1及pLP10L-TF2/pLP10L-TR2对平板上的克隆进行PCR筛选，正确阳性克隆PCR产物分别长1065bp、537bp。PCR片段再进行测序验证，共同确定正确的克隆，该正确克隆中含有连接产物-质粒pLP10。扩大培养正确克隆，并提取质粒pLP10。质粒pLP10的多克隆位点AhdI-EcoRI-SacI-AhdI-NheI，分别通过在引物pSW23T-F及pSW25T-F的5’端 上引入酶切位点形成。
(5)以质粒pLP10为模板，采用引物pLP10-F/pLP10-R进行反向PCR扩增，获得片段pLP10-1，以去掉pLP10中完整的ccdB基因。以大肠杆菌LP79(构建方法参考发明人文章，Carraro N,Matteau D,Luo P,et al.PLOS Genetics,2014,10(10):e1004714)基因组为模板，采用vmi480-F/vmi480-R进行PCR扩增，获得完整的vmi480基因片段，其核苷酸序列如SEQ ID NO.1的第21-617位碱基序列所示。大肠杆菌LP79事先通过接合转移获得了来自拟态弧菌VM573的基因岛MGIVmi1,该基因岛含有vmi480致死基因。
(6)PCR片段pLP10-1与vmi480基因片段分别进行NdeI/XhoI双酶切并纯化后，以T4DNA连接酶进行连接，连接产物(质粒pLP11)转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以pLP11L-TF/pLP11L-TR引物对平板上的克隆进行PCR筛选，正确克隆阳性PCR产物长515bp，PCR片段再进行测序验证，共同确定正确的克隆，该正确克隆中含有连接产物-质粒pLP11。扩大培养正确克隆，并提取质粒pLP11。
(7)以质粒pLP11为模板，采用引物pLP11-F/pLP11-R进行反向PCR扩增，获得片段pLP11-1，以去掉pLP11完整的PTAC启动子系统。以pBAD30质粒(普如汀生物,NTCC 1809)为模板，采用引物pBAD30-PF/pBAD30-PR为引物扩增，获得pBAD30质粒中PBAD启动子部分，命名为扩增片段pBAD30-1，扩增片段pBAD30-1的两端各含有一段与pLP11-1两端相同的序列。采用无缝克隆试剂盒(Vazyme)对pLP11-1和pBAD30-1进行体外重组，重组产物(质粒pLP12)转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以pLP12L-TF/pLP12L-TR引物对平板上的克隆进行PCR筛选，正确阳性克隆PCR产物长1104bp，PCR片段再进行测序验证，共同确定正确的克隆，该正确克隆中含有重组产物-质粒pLP12。扩大培养正确的克隆，并提取质粒pLP12，即为弧菌通用的基因敲除自杀载体pLP12，其核苷酸序列如SEQ ID NO.1所示。具体结构如图1所示。
(8)以弧菌通用的基因敲除自杀载体pLP12为模板，再进行PCR扩增，扩增引物STVU-F/STVU-R。扩增产物经PCR产物纯化试剂盒纯化后进行AhdI酶切，酶切后采用DNA浓缩试剂盒进行纯化(中鼎)，获得线性化的弧菌通用的基因敲除自杀载体pLP12T(其核苷酸序列如SEQ ID NO.2所示)。pLP12T双链的3’端各有一个突出的碱基T,因此可以与普通DNA聚合酶扩增产生PCR产物直接连接。二者都不须再进行酶切。
实施例2应用pLP12T敲除溶藻弧菌溶血素基因(vah)
(1)以交叠PCR方式扩增得到溶藻弧菌E0601的vah基因框内缺失融合片段。第一轮PCR引物对分别为vah-MF1/vah-MR1、vah-MF2/vah-MR2，模板为溶藻弧菌E0601基因组DNA。第一轮分别产生PCR产物vah1、vah2。vah1、vah2经PCR产物纯化试剂盒(Axygen)纯化后,等体积混合作为第二轮PCR的模板。第二轮PCR采用引物vah-MF1/vah-MR2，采用普通Taq DNA聚合酶(TaKaRa)，PCR扩增条件PCR扩增条件参考酶说明书，由此得到第二轮PCR产物。
(2)第二轮PCR产物经纯化后，直接与上述弧菌通用的基因敲除自杀载体pLP12T进行连接，采用T4DNA连接酶。连接产物(质粒pLP12-vah)直接转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以vah-MF1/vah-MR2引物对平板上的克隆进行PCR筛选，正确的克隆会产生长660bp扩增条带。PCR片段再进行测序验证，共同确定正确的克隆，该正确的克隆含有连接产物-质粒pLP12-vah。扩大培养正确克隆，并提取质粒pLP12-vah。
(3)pLP12-vah电转化大肠杆菌β2163细胞(现有技术中的菌株，构建方法Demarre G,et al.A new family of mobilizable suicide plasmids based on broad host range R388plasmid(IncW)and RP4plasmid(IncP alpha)conjugative machineries and their cognate Escherichia coli host strains.Research in Microbiology,2005,156:245-255)。电转化条件：电压1800V，电击时间5ms。电击后细胞于37℃恢复培养1hr，涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。过夜培养，挑取任意克隆，纯化并保存，即得到包含自杀质粒pLP12-vah的供体菌—大肠杆菌β2163(pLP12-vah)。
(4)过夜培养大肠杆菌β2163(pLP12-vah)以及受体菌溶藻弧菌E0601。取100μL供体菌与100μL受体菌混合，8000g离心2min弃上清，加入400μL新鲜LB培养基重悬，再次8000g离心2min小心弃去上清。加入10μL新鲜LB培养基重悬，将重悬液全部吸入并滴在LB琼脂平板上。超净台风干后，37℃倒置培养6hr。
(5)以1ml新鲜LB培养基充分吹悬上述培养物，取100μL涂布LB琼脂平板(含0.3％D-葡萄糖、10μg/ml氯霉素)，37℃过夜培养。在此平板上，只有发生第一次同源重组，染色体整合有自杀质粒pLP12-vah的插入突变溶藻弧菌细胞可以生长。随机挑取克隆进行PCR鉴定，PCR引物vah-MF1/vah-MR2，正确的克隆产生1218bp和660bp二个条带(图3中的泳道2)，得到vah插入突变株。
(6)vah插入突变株于LB(0.3％D-葡萄糖)培养3小时后，梯度稀释，各取100μL涂布LB琼脂平板(0.2％L-阿拉伯糖)，37℃过夜培养。在此平板上，只有发生第二次同源重 组，染色体丢失整合态自杀质粒pLP12-vah的细胞能够存活。细菌细胞发生第二次重组后，会产生一个vah基因缺失的细胞和一个包含野生型vah基因的细胞。
(7)以vah-MF1/vah-MR2引物对LB琼脂平板(0.2％L-阿拉伯糖)上的克隆进行PCR检测，正确的vah基因缺失克隆产生660bp的条带(图3中的泳道3)，第二次重组形成的野生型克隆产生1218bp条带(图3中的泳道1)。正确的vah基因缺失克隆再经PCR产物测序验证，即得到vah基因缺失株，从而完成溶藻弧菌vah基因敲除。本实施例中，反向选择平板(10-1稀释)上随机挑取的16个克隆中，有12个为正确的vah基因缺失克隆。
实施例3应用pLP12T敲除霍乱弧菌周质丝氨酸肽酶基因(degS)
(1)以交叠PCR扩增得到霍乱弧菌HN375的degS基因框内缺失融合片段。第一轮PCR引物对分别为degs-MF1/degs-MR1、degs-MF2/degs-MR2，模板为霍乱弧菌HN375基因组DNA。两个PCR产物等体积混合后纯化，作为第二轮PCR的模板。第二轮PCR采用引物degs-MF1/degs-MR2，采用普通Taq DNA聚合酶，得到第二轮PCR产物。
(2)上述第二轮PCR产物，直接与自杀载体pLP12T进行连接。连接产物(质粒pLP12-degS)转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以degs-MF1/degs-MR2引物对平板上的克隆进行PCR筛选，正确的克隆会产生长926bp扩增条带。PCR片段再进行测序验证，共同确定正确的克隆，该正确的克隆中含有连接产物-质粒pLP12-degS。扩大培养正确克隆，并提取质粒pLP12-degS。
(3)pLP12-degS电转化大肠杆菌β2163细胞。电转化条件及电击后细胞培养条件同实施例2。挑取任意克隆，纯化并保存，即得到包含自杀质粒pLP12-degS的供体菌—大肠杆菌β2163(pLP12-degS)。
(4)过夜培养大肠杆菌β2163(pLP12-degS)以及受体菌霍乱弧菌HN375。取100μL供体菌与100μL受体菌混合，8000g离心3min弃上清，加入500μL新鲜LB培养基重悬，再次8000g离心2min小心弃去上清。加入10μL新鲜LB培养基重悬，将重悬液全部吸入并滴在LB琼脂平板上。超净台风干后，37℃倒置培养7hr。
(5)以1ml新鲜LB培养基充分吹悬上述培养物，取100μL涂布LB琼脂平板(含10μg/ml氯霉素、0.3％D-葡萄糖)，37℃过夜培养。在此平板上，只有发生第一次同源重组，染色体整合有自杀质粒pLP12-degS的插入突变霍乱弧菌细胞才可以生长。随机挑取克隆进行PCR鉴定，PCR引物degS-MF1/degS-MR2，正确的克隆产生1496bp和926bp二个条带(图3中的泳道5)，即得到degS插入突变株。
(6)degS插入突变株于LB(0.3％D-葡萄糖)培养3小时后，梯度稀释，各取100μL涂布LB琼脂平板(0.2％L-阿拉伯糖)，37℃过夜培养。在此平板上，只有发生第二次同源重组，染色体丢失整合态自杀质粒pLP12-degS的细胞能够存活。细菌细胞发生第二次重组后，会产生一个degS基因缺失的细胞和一个包含野生型degS基因的细胞。
(7)以degS-MF1/degS-MR2引物对LB琼脂平板(0.2％L-阿拉伯糖)上的克隆进行PCR检测，正确的degS基因缺失克隆产生926bp的条带(图3中的泳道6)，第二次重组形成的野生型克隆产生1496bp条带(图3中的泳道4)。正确的degS基因缺失克隆再经PCR产物测序验证，即得到degS基因缺失株，从而完成霍乱弧菌degS基因敲除。本实施例中，反向选择平板(10-1稀释)上随机挑取的16个克隆中，有8个为正确的degS基因缺失克隆。
实施例4应用pLP12T敲除霍乱弧菌VI型分泌系统基因(vasC)
(1)以交叠PCR扩增得到霍乱弧菌HN375的vasC基因框内缺失融合片段。第一轮PCR引物对分别为vasC-MF1/vasC-MR1、vasC-MF2/vasC-MR2，模板为霍乱弧菌HN375基因组DNA。两个PCR产物纯化后,等体积混合作为第二轮PCR的模板。第二轮PCR引物vasC-MF1/vasC-MR2，采用普通Taq DNA聚合酶，得到第二轮PCR产物。
(2)第二轮PCR产物经纯化后，直接与实施例1制备的基因敲除自杀载体pLP12T进行连接。连接产物(质粒pLP12-vasC)转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以vasC-MF1/vasC-MR2引物对平板上的克隆进行PCR筛选，正确的克隆会产生长925bp扩增条带。PCR片段再进行测序验证，共同确定正确的克隆，该正确克隆含有连接产物(质粒pLP12-vasC)。扩大培养正确克隆，并提取质粒pLP12-vasC。
(3)pLP12-vasC电转化大肠杆菌β2163细胞。电转化条件及电击后细胞培养条件同实施例2。挑取任意克隆，纯化并保存，即得到包含自杀质粒pLP12-vasC的供体菌—大肠杆菌β2163(pLP12-vasC)。
(4)过夜培养大肠杆菌β2163(pLP12-vasC)以及受体菌霍乱弧菌HN375。取100μL供体菌与100μL受体菌混合，8000g离心2min弃上清，加入600μL新鲜LB培养基重悬，再次8000g离心2min，小心弃去上清。加入10μL新鲜LB培养基重悬，将重悬液全部吸入并滴在LB琼脂平板上。超净台风干后，37℃倒置培养8hr。
(5)以1ml新鲜LB培养基充分吹悬上述培养物，取100μL涂布LB琼脂平板(含0.3％D-葡萄糖、10μg/ml氯霉素)，37℃过夜培养。在此平板上，只有发生第一次同源重组，染 色体整合有自杀质粒pLP12-vasC的插入突变霍乱弧菌细胞可以生长。随机挑取克隆进行PCR鉴定，PCR引物vasC-MF1/vasC-MR2，正确的克隆产生1630bp和925bp二个条带(图3中的泳道8)，得到vasC插入突变株。
(6)vasC插入突变株于LB(0.3％D-葡萄糖)培养3小时后，梯度稀释，各取100μL涂布LB琼脂平板(0.2％L-阿拉伯糖)，37℃过夜培养。在此平板上，只有发生第二次同源重组，染色体丢失整合态自杀质粒pLP12-vasC的细胞能够存活。细菌细胞经第二次重组产生一个vasC基因缺失的细胞和一个包含野生型vasC基因的细胞。
(7)以vasC-MF1/vasC-MR2引物对LB琼脂平板(0.2％L-阿拉伯糖)上的克隆进行PCR检测，正确的vasC基因缺失克隆产生925bp的条带(图3中的泳道9)，第二次重组形成的野生型克隆则产生1630bp条带(图3中的泳道7)。正确的vasC基因缺失克隆再经PCR产物测序验证，即得到vasC基因缺失株，从而完成霍乱弧菌vasC基因敲除。本实施例中，反向选择平板(10-1稀释)上随机挑取的16个克隆中，有7个为正确的vasC基因缺失克隆。
实施例5应用pLP12T敲除副溶血弧菌IV型鞭毛合成基因(pilO)
(1)以交叠PCR扩增得到副溶血弧菌E0680的pilO基因框内缺失融合片段。第一轮PCR引物对分别为pilO-MF1/pilO-MR1、pilO-MF2/pilO-MR2，模板为副溶血弧菌E0680基因组DNA。两个PCR产物纯化后,等体积混合作为第二轮PCR模板。第二轮PCR引物pilO-MF1/pilO–MR2，采用普通Taq DNA聚合酶。得到第二轮PCR产物。
(2)第二轮PCR产物经纯化后，直接与弧菌通用的基因敲除自杀载体pLP12T进行连接。连接产物(质粒pLP12-pilO)转化大肠杆菌DH5αλpir细胞，涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以pilO-MF1/pilO–MR2引物对平板上的克隆进行PCR筛选，正确的克隆会产生大小为815bp的扩增条带。PCR片段再进行测序验证，共同确定正确的克隆，该正确克隆含有连接产物-质粒pLP12-pilO。扩大培养正确克隆，并提取质粒pLP12-pilO。
(3)pLP12-pilO电转化大肠杆菌β2163细胞。电转化条件及电击后细胞培养条件同实施例2。挑取任意克隆，纯化并保存，即得到包含自杀质粒pLP12-pilO的供体菌—大肠杆菌β2163(pLP12-pilO)。
(4)过夜培养大肠杆菌β2163(pLP12-pilO)以及受体菌副溶血弧菌E0680。取100μL供体菌与100μL受体菌混合，8000g离心2min弃上清，加入400μL新鲜LB培养基重悬，再次8000g离心2min，小心弃去上清。加入10μL新鲜LB培养基重悬，将重悬液全部吸 入并滴在LB琼脂平板上。超净台风干后，37℃倒置培养8hr。
(5)以1ml新鲜LB培养基充分吹悬上述培养物，取100μL涂布LB琼脂平板(含0.3％D-葡萄糖、10μg/ml氯霉素)，37℃过夜培养。在此平板上，只有发生第一次同源重组，染色体整合有自杀质粒pLP12-pilO的插入突变副溶血弧菌细胞才可以生长。随机挑取克隆进行PCR鉴定，PCR引物pilO-MF1/pilO–MR2，正确的克隆产生1214bp和815bp二个条带(图3中的泳道11)，得到pilO插入突变株。
(6)pilO插入突变株于LB(0.3％D-葡萄糖)培养3小时后，梯度稀释，各取100μL涂布LB琼脂平板(0.2％L-阿拉伯糖)，37℃过夜培养。在此平板上，只有发生第二次同源重组，染色体丢失整合态自杀质粒pLP12-pilO的细胞才能够存活。细菌细胞发生第二次重组会产生一个pilO基因缺失的细胞和一个包含野生型pilO基因的细胞。
(7)以pilO-MF1/pilO-MR2引物对LB琼脂平板(0.2％L-阿拉伯糖)上的克隆进行PCR检测，正确的pilO基因缺失克隆产生815bp的条带(图3中的泳道12)，第二次重组形成的野生型克隆产生1214bp条带(图3中的泳道10)。正确的pilO基因缺失克隆再经PCR产物测序验证，即得到pilO基因缺失株，从而完成副溶血弧菌pilO基因敲除。本实施例中，反向选择平板(10-1稀释)上随机挑取的16个克隆中，有7个为正确的pilO基因缺失克隆。
实施例6应用pLP12T敲除副溶血弧菌前蛋白移位酶基因(ascS)
(1)以交叠PCR扩增副溶血弧菌E06135的ascS基因，得到ascS框内缺失融合片段。第一轮PCR引物对分别为ascS-MF1/ascS-MR1、ascS-MF2/ascS-MR2，模板为副溶血弧菌E06135基因组DNA。两个PCR产物等体积混合并纯化后作为第二轮PCR的模板。第二轮PCR引物ascS-MF1/ascS–MR2，采用普通Taq DNA聚合酶，得到第二轮PCR产物。
(2)将第二轮PCR产物经纯化后，直接与上述弧菌通用的基因敲除自杀载体pLP12T进行连接。连接产物(质粒pLP12-ascS)转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以ascS-MF1/ascS–MR2引物对平板上的克隆进行PCR筛选，正确的克隆会产生703bp的扩增条带。PCR片段再进行测序验证，共同确定正确的克隆，该正确克隆含有连接产物-质粒pLP12-ascS。扩大培养正确克隆，并提取质粒pLP12-ascS。
(3)pLP12-ascS电转化大肠杆菌β2163细胞。电转化条件及电击后细胞培养条件同实施例2。挑取任意克隆，纯化并保存，即得到包含自杀质粒pLP12-ascS的供体菌—大肠杆菌β2163(pLP12-ascS)。
(4)过夜培养大肠杆菌β2163(pLP12-ascS)以及受体菌副溶血弧菌E06135。取100μL供体菌与100μL受体菌混合，8000g离心2min弃上清，加入400μL新鲜LB培养基重悬，再次8000g离心2min小心弃去上清。加入10μL新鲜LB培养基重悬，将重悬液全部吸入并滴在LB琼脂平板上。超净台风干后，37℃倒置培养8hr。
(5)以1ml新鲜LB培养基充分吹悬上述培养物，取100μL涂布LB琼脂平板(含0.3％D-葡萄糖、10μg/ml氯霉素)，37℃过夜培养。在此平板上，只有发生第一次同源重组，染色体整合有自杀质粒pLP12-ascS的插入突变副溶血弧菌细胞才可以生长。随机挑取克隆进行PCR鉴定，PCR引物ascS-MF1/ascS–MR2，正确的克隆产生952bp和703bp二个条带(图3中的泳道14)，得到ascS插入突变株。
(6)ascS插入突变株于LB(0.3％D-葡萄糖)培养4小时，梯度稀释，各取100μL涂布LB琼脂平板(0.2％L-阿拉伯糖)，37℃过夜培养。在此平板上，只有发生第二次同源重组，染色体丢失整合态自杀质粒pLP12-degS的细胞才能够存活。细菌细胞发生第二次重组后，会产生一个ascS基因缺失的细胞和一个包含野生型ascS基因的细胞。
(7)以ascS-MF1/ascS-MR2引物对LB琼脂平板(0.2％L-阿拉伯糖)上的克隆进行PCR检测，正确的ascS基因缺失克隆产生703bp的条带(图3中的泳道15)，第二次重组形成的野生型克隆产生952bp条带(图3中的泳道13)。正确的ascS基因缺失克隆再经PCR产物测序验证，即得到ascS基因缺失株，从而完成副溶血弧菌ascS基因敲除。本实施例中，反向选择平板(10-1稀释)上随机挑取的16个克隆中，有3个为正确的ascS基因缺失克隆。
实施例7应用pLP12T敲除创伤弧菌VI型分泌系统基因(impB)
(1)以交叠PCR扩增得到创伤弧菌ATCC 27562的impB基因框内缺失融合片段。第一轮PCR引物对分别为impB-MF1/impB-MR1、impB-MF2/impB-MR2，模板为创伤弧菌ATCC27562基因组DNA。两个PCR产物纯化后,等体积混合作为第二轮PCR的模板。第二轮PCR采用引物impB-MF1/impB–MR2，采用普通Taq DNA聚合酶，得到第二轮PCR产物。
(2)第二轮PCR产物经纯化后，直接与实施例1制备的弧菌通用的基因敲除自杀载体pLP12T进行连接。连接产物(质粒pLP12-impB)直接转化大肠杆菌DH5αλpir细胞，并涂布LB平板(含20μg/ml氯霉素、0.3％D-葡萄糖)。以impB-MF1/impB-MR2引物对平板上的克隆进行PCR筛选，正确的克隆会产生长909bp扩增条带。PCR片段再进行测序验证，共同确定正确的克隆，该正确克隆含有连接产物-质粒pLP12-impB。扩大培养正确克隆，并提取质粒pLP12-impB。
(3)pLP12-impB电转化大肠杆菌β2163细胞。电转化条件及电击后细胞培养条件同实施例2。过夜培养，挑取任意克隆，纯化并保存，即得到包含自杀质粒pLP12-impB的供体菌—大肠杆菌β2163(pLP12-impB)。
(4)过夜培养大肠杆菌β2163(pLP12-impB)以及受体菌创伤弧菌ATCC 27562。取100μL供体菌与100μL受体菌混合，8000g离心3min弃上清，加入600μL新鲜LB培养基重悬，再次8000g离心3min，小心弃去上清。加入10μL新鲜LB培养基重悬，将重悬液全部吸入并滴在LB琼脂平板上。超净台风干后，37℃倒置培养7hr。
(5)以1ml新鲜LB培养基充分吹悬上述培养物，取100μL涂布LB琼脂平板(含0.3％D-葡萄糖、10μg/ml氯霉素)，37℃过夜培养。在此平板上，只有发生第一次同源重组后染色体整合有自杀质粒pLP12-impB的插入突变创伤弧菌细胞才可以生长。随机挑取克隆进行PCR鉴定，PCR引物impB-MF1/impB-MR2，正确的克隆产生1290bp和909bp二个条带(图3中的泳道17)，得到impB插入突变株。
(6)impB插入突变株于LB(0.3％D-葡萄糖)培养3小时，梯度稀释，各取100μL涂布LB琼脂平板(0.2％L-阿拉伯糖)，37℃过夜培养。在此平板上，只有发生第二次同源重组，染色体丢失整合态自杀质粒pLP12-impB的细胞能够存活。细菌细胞发生第二次重组后，会产生一个impB基因缺失的细胞和一个包含野生型impB基因的细胞。
(7)以impB-MF1/impB-MR2引物对LB琼脂平板(0.2％L-阿拉伯糖)上的克隆进行PCR检测，正确的impB基因缺失克隆产生909bp的条带(图3中的泳道18)，第二次重组形成的野生型克隆则产生1290bp条带(图3中的泳道16)。正确的impB基因缺失克隆再经PCR产物测序验证，即得到impB基因缺失株，从而完成副溶血弧菌impB基因敲除。本实施例中，反向选择平板(10-1稀释)上随机挑取的20个克隆中，有6个为正确的impB基因缺失克隆。
上述实施例2-7中的反向选择平板均有NaCl的存在，然而缺失突变株仍然有很高的出现比率，说明致死基因发挥了作用，且不受NaCl存在的影响。
Figure PCTCN2015097202-appb-000004
Figure PCTCN2015097202-appb-000005
Figure PCTCN2015097202-appb-000006
Figure PCTCN2015097202-appb-000007
Figure PCTCN2015097202-appb-000008
Figure PCTCN2015097202-appb-000009
Figure PCTCN2015097202-appb-000010

Claims (8)

  1. 一种基因敲除自杀载体pLP12，为环状载体，其特征在于，包括PBAD启动子、阻遏蛋白基因araC、RP4转移起始位点、氯霉素抗性基因、R6K复制起始位点、多克隆位点区和致死基因vmi480，所述的多克隆位点区至少包含两个AhdI酶切位点，所述的致死基因vmi480的核苷酸序列如SEQ ID NO.1的第21-617位碱基序列所示，所述的致死基因vmi480位于PBAD启动子下游并处于PBAD启动子控制之下。
  2. 根据权利要求1所述的基因敲除自杀载体pLP12，其特征在于，所述的基因敲除自杀载体pLP12，其核苷酸序列如SEQ ID NO.1所示。
  3. 一种基因敲除自杀载体pLP12T，为线性载体，其特征在于，是将权利要求1所述的基因敲除自杀载体pLP12用AhdI酶切多克隆位点区中的两个AhdI酶切位点后得到的线性载体，即为基因敲除自杀载体pLP12T。
  4. 根据权利要求3所述的基因敲除自杀载体pLP12T，其特征在于，所述的基因敲除自杀载体pLP12T，其核苷酸序列如SEQ ID NO.2所示。
  5. 权利要求3所述的基因敲除自杀载体pLP12T在基因敲除中的应用。
  6. 根据权利要求5所述的应用，其特征在于，所述的基因敲除自杀载体pLP12T在弧菌基因敲除中的应用。
  7. 根据权利要求5所述的应用，其特征在于，将目的基因的框内缺失融合片段与基因敲除自杀载体pLP12T连接，得到携带目的基因的框内缺失融合片段的pLP12T，命名为pLP12T-X，pLP12T-X经转化进入供体菌中，然后将此供体菌与受体菌混合，经接合作用将pLP12T-X转移进入受体菌，经氯霉素选择，获得pLP12T-X整合到受体菌基因组靶位点的目的基因插入突变型菌株，然后此目的基因插入突变型菌株在阿拉伯糖诱导vmi480基因产生毒素Vmi480的反向选择压力下，经过筛选获得目的基因缺失的克隆，从而达到将目的基因敲除，获得目的基因缺失的菌株。
  8. 根据权利要求7所述的应用，其特征在于，所述的受体菌为弧菌。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109880844A (zh) * 2019-03-06 2019-06-14 江苏省农业科学院 一种基于同源重组的变棕溶杆菌oh23基因敲除系统的构建方法与应用
CN110591993A (zh) * 2019-09-09 2019-12-20 中国水产科学研究院南海水产研究所 一种基于盐酸刺激的哈维弧菌同源重组基因敲除的方法

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105063073A (zh) * 2015-09-16 2015-11-18 中国科学院南海海洋研究所 一种弧菌通用的基因敲除自杀载体及其应用
CN105505974B (zh) * 2016-01-21 2019-01-11 浙江大学 一种实现大肠杆菌基因无痕同源重组的方法
CN106399213A (zh) * 2016-08-09 2017-02-15 江苏省农业科学院 一种抗生素溶杆菌基因敲除系统及其构建方法和应用
CN106834332B (zh) * 2017-03-09 2021-05-04 中国科学院过程工程研究所 一种重组质粒及其制备方法和应用
CN109517844B (zh) * 2018-10-17 2021-04-30 中国水产科学研究院东海水产研究所 一种鳗弧菌鞭毛蛋白基因敲除的方法
CN110066820B (zh) * 2019-04-29 2021-03-16 华南农业大学 一种荧光菌株E.coli C600及构建方法与应用
CN110591994B (zh) * 2019-09-09 2023-10-20 中国水产科学研究院南海水产研究所 一种基于氢氧化钠刺激的哈维弧菌同源重组基因敲除的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104817632A (zh) * 2015-05-05 2015-08-05 中国科学院南海海洋研究所 拟态弧菌ta系统毒素蛋白及其编码基因和应用
CN105063073A (zh) * 2015-09-16 2015-11-18 中国科学院南海海洋研究所 一种弧菌通用的基因敲除自杀载体及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0121998D0 (en) * 2001-09-11 2001-10-31 Acambis Res Ltd Attenuated bacteria useful in vaccines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104817632A (zh) * 2015-05-05 2015-08-05 中国科学院南海海洋研究所 拟态弧菌ta系统毒素蛋白及其编码基因和应用
CN105063073A (zh) * 2015-09-16 2015-11-18 中国科学院南海海洋研究所 一种弧菌通用的基因敲除自杀载体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LUO, PENG ET AL.: "Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480", PLOS ONE, vol. 10, no. 12, 7 December 2015 (2015-12-07), pages 1 - 17, XP055373177, ISSN: 1932-6203 *
ROUX, F.L. ET AL.: "Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 73, no. 3, 28 February 2007 (2007-02-28), pages 777 - 784, XP002674498, ISSN: 0099-2240 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109880844A (zh) * 2019-03-06 2019-06-14 江苏省农业科学院 一种基于同源重组的变棕溶杆菌oh23基因敲除系统的构建方法与应用
CN110591993A (zh) * 2019-09-09 2019-12-20 中国水产科学研究院南海水产研究所 一种基于盐酸刺激的哈维弧菌同源重组基因敲除的方法

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