CN118291354A - Streptococcus agalactiae luminous bacteria based on luciferase reporting system and construction method thereof - Google Patents
Streptococcus agalactiae luminous bacteria based on luciferase reporting system and construction method thereof Download PDFInfo
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Abstract
The invention discloses streptococcus agalactiae luminous bacteria based on a luciferase reporting system and a construction method thereof. The invention provides a plasmid, which is obtained by seamless cloning of pSET4s vector skeleton, P23 promoter sequence and sacB gene; the nucleotide sequence is sequence 8. The invention constructs a plasmid for expressing a luciferase reporting system, and transfers the plasmid into streptococcus agalactiae to obtain streptococcus agalactiae luminous bacteria, wherein the streptococcus agalactiae luminous bacteria are obtained by traceless insertion of luciferase and a promoter thereof into a streptococcus agalactiae genome, the screening pressure of antibiotics is not needed, a luciferase gene can be replicated and expressed constitutively along with the genome of pathogenic bacteria, the infection and pathogenic process of pathogenic bacteria are reduced most truly, and the strain can be observed in real time in an animal body.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to streptococcus agalactiae luminous bacteria based on a luciferase reporting system and a construction method thereof.
Background
Streptococcus agalactiae, also known as group B streptococcus, is an important gram-positive pathogenic bacterium, named after its first separation in cows suffering from mastitis. Milk chain free globules are the primary causative agent of neonatal meningitis and sepsis, and also cause invasive infections in adults, an important bacterium that causes global burden. Therefore, the research on the pathogenic mechanism of the infection path of streptococcus agalactiae and the related virulence factors is promoted, and theoretical support is provided for preventing and treating the streptococcus agalactiae. The trace technology based on bioluminescence can help to know the space-time variation of pathogens in animals, and trace observation of streptococcus agalactiae is reported to be realized based on luciferase reporter gene plasmid, but the selection pressure of antibiotics is required to maintain the existence of the plasmid, so that the real situation of streptococcus agalactiae in animals cannot be reduced.
The luciferase reporter gene utilizes firefly luciferase and the like to catalyze substrate luciferin oxidation reaction to carry out bioluminescence in the presence of ATP and oxygen, has the advantages of high sensitivity, strong specificity, no autofluorescence, capability of penetrating tissues, accurate quantification and the like, and presents potential application value in pathogen pathogenesis research. The levansucrase encoded by the sacB gene of bacillus subtilis can polymerize sucrose to convert into a levan polymer, while in other bacteria, in the presence of sucrose, the expression of the sacB gene can cause accumulation of the levan polymer in cells or outside cells, and damage normal cellular processes to cause lethal damage, so that the sensitivity of sucrose produced by sacB expression can be used as a reverse screen for genetic manipulation.
Disclosure of Invention
The invention solves the technical problem of how to observe streptococcus agalactiae in real time in an animal body.
In order to solve the technical problems, the first aspect of the invention provides streptococcus agalactiae luminous bacteria, which is a bacterial strain obtained by inserting fragments consisting of a target luminous gene promoter and a target luminous gene into streptococcus agalactiae genome in a traceless way.
The above traceless insertion is an insertion without a selectable marker or other fragment.
The above-mentioned position of traceless insertion is between SAG2025 and SAG2026 genes of Streptococcus agalactiae 2603V/R (GenBank: AE009948, 31-JAN-2014) genome, at 2003475.
In an embodiment of the present invention, streptococcus agalactiae light emitting bacteria may be a fragment consisting of the CP25 promoter and the Luc gene (nucleotide sequence consisting of sequence 6 and sequence 7) inserted between SAG2025 and SAG2026 genes of Streptococcus agalactiae 2603V/R (GenBank: AE009948, 31-JAN-2014) genome at 2003475.
In a second aspect, the present invention provides a method for preparing the streptococcus agalactiae luminous bacteria of the first aspect, comprising the steps of: the recombinant plasmid is led into streptococcus agalactiae, and then homologous recombination and reverse screening marker sacB identification are carried out to obtain streptococcus agalactiae luminous bacteria;
The recombinant plasmid is obtained by inserting a fragment containing a target luminous gene promoter and a target luminous gene into a shuttle plasmid;
The shuttle plasmid comprises a pSET4s vector skeleton, sacB genes and a promoter for driving the expression of the sacB genes;
the nucleotide sequence of the pSET4s vector framework is a sequence 3;
the nucleotide sequence of the sacB gene is sequence 2.
In the method, the promoter for driving the sacB gene expression is a P23 promoter, and the nucleotide sequence of the promoter is a sequence 1;
The nucleotide sequence of the shuttle plasmid is sequence 8.
In the above method, the fragment containing the target light-emitting gene promoter and the target light-emitting gene comprises an upstream homology arm for homologous recombination into the streptococcus agalactiae genome, the target light-emitting gene promoter, the target light-emitting gene and the downstream homology arm for homologous recombination into the streptococcus agalactiae genome.
In the above method, the target light emitting gene promoter is a CP25 promoter.
The nucleotide sequence of the CP25 promoter may be sequence 6;
The target luminescent gene is Luc gene.
The nucleotide sequence of the Luc gene may be the sequence 7.
Further, the nucleotide sequence for homologous recombination into the upstream homology arm of the streptococcus agalactiae genome may be sequence 4;
the nucleotide sequence for homologous recombination into the downstream homology arm of the streptococcus agalactiae genome may be sequence 5.
The method specifically comprises the following steps:
1) The recombinant plasmid is introduced into streptococcus agalactiae to obtain transformed bacteria;
2) Culturing the transformed bacteria in a resistance culture medium, selecting ID-F (corresponding to fragments on a genome) and CP25-R (corresponding to detection of a promoter CP 25) as primers for identification, and selecting a strain with 1095bp amplification product to obtain a strain with upstream homology arm exchange;
3) And subculturing the strain subjected to upstream homology arm exchange in a culture medium without antibiotics at 28 ℃, coating the strain on a solid THB plate without antibiotics and containing 0.75M sucrose after the strain grows to a plateau, selecting the grown strain for identification by taking ID-F (corresponding to fragments on a genome) and ID-R (corresponding to fragments on the genome) as primers, and selecting the strain with 3592bp amplification product to obtain the streptococcus agalactiae luminous bacteria.
In a third aspect, streptococcus agalactiae light-emitting bacteria prepared by the method of the second aspect.
In a fourth aspect, the present invention provides the use of a streptococcus agalactiae light-emitting bacterium as described in the first or third aspect or the method as described in the second aspect for the preparation of a streptococcus agalactiae infected cell model or an animal model of streptococcus agalactiae infection;
or the invention provides the use of a streptococcus agalactiae light-emitting bacterium as described in the first or third aspect or a method as described in the second aspect in the manufacture of a product for in vivo streptococcus agalactiae tracer observation in an animal.
In a fifth aspect, the present invention provides a streptococcus agalactiae tracer observation product for use in animals comprising a luminescent streptococcus agalactiae according to the first or third aspect.
In a sixth aspect, the present invention provides a shuttle plasmid as described in the second aspect or the recombinant plasmid.
In a seventh aspect, the present invention provides the use of a shuttle plasmid according to the second aspect or of a recombinant plasmid according to any one of the following:
1) Preparing a streptococcus agalactiae infection cell model or an animal model of streptococcus agalactiae infection;
2) The streptococcus agalactiae tracing and observing product in animals is prepared.
The invention inserts the luciferase gene and the constitutive expression promoter into the genome of the pathogenic bacteria through homologous recombination, and the pathogenic bacteria capable of bioluminescence are constructed tracelessly by using a reverse screening method, so that the luciferase gene can replicate and constitutively express along with the genome of the pathogenic bacteria without depending on the screening pressure of antibiotics, and the infection and pathogenic process of the pathogenic bacteria are reduced most truly.
Experiments prove that the invention constructs a plasmid for expressing a luciferase reporting system, and transfers the plasmid into streptococcus agalactiae to obtain streptococcus agalactiae luminous bacteria, wherein the streptococcus agalactiae luminous bacteria are obtained by inserting luciferases and promoters thereof into streptococcus agalactiae genome in a traceless manner, the screening pressure of antibiotics is not needed, and the luciferase genes can be replicated and expressed constitutively along with pathogen genome, so that the infection and pathogenic process of pathogenic bacteria can be reduced in the truest, and the strain can be observed in real time in animals.
Drawings
FIG. 1 shows the results of PCR detection of pSET4s vector backbone, P23 promoter and sacB gene.
FIG. 2 shows the results of the detection of the overlapping PCR products of the upstream homology arm 2025, downstream homology arm 2026 and CP 25-Luc.
FIG. 3 is a PCR identification of a monoclonal.
FIG. 4 is an electrophoretogram of screening for single exchange of upstream homology arms.
FIG. 5 is an electrophoretogram identifying light-emitting Streptococcus agalactiae.
FIG. 6 shows the luminescence intensity of bacteria detected by the luminescence detector.
FIG. 7 shows the results of detection of the luminescence type Streptococcus agalactiae infection in real time in animals.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 preparation of Streptococcus agalactiae light-emitting bacteria
1. Construction of Streptococcus agalactiae luminous bacteria
1. Construction pKSAB E.coli-Streptococcus agalactiae shuttle plasmid
Using Gloria Nova HS X Master Mix (abclon a, RK 20717) PCR reaction system, amplifying the pSET4S vector backbone (sequence 3) with 4S-F/4S-R as primers and pSET4S as template; amplifying the P23 promoter sequence (sequence 1) by using the P23-F/P23-R as a primer and using a synthesized P23 promoter sequence plasmid as a template; the levansucrase sacB gene (SEQ ID NO: 2) was amplified using sacB-F/sacB-R as a primer and pCasPA (WU.S. vast, biotechnology Co., ltd., P23627) plasmid as a template.
The PCR conditions were as follows:
Pre-denaturation at 98℃for 45s
Denaturation at 98℃for 10s
Annealing at 60℃for 20s for 30 cycles
15S/kb extension at 72 ℃
Final extension at 72℃for 5min
As a result of agarose gel electrophoresis of the PCR product, the pSET4s vector has a skeleton of 4418bp, a P23 promoter (shown as P23 promoter) of 312bp and a sacB gene of 1422bp as shown in FIG. 1. The PCR products were recovered using a common DNA product recovery kit (Tiangen Biochemical Co., ltd., DP 204).
The pSET4s vector backbone (SEQ ID NO: 3), P23 promoter sequence (SEQ ID NO: 1) and sacB gene fragment (SEQ ID NO: 2) obtained as described above were subjected to seamless cloning by incubating 20 min at 50℃with 2X MultiF Seamless Assembly Mix (ABclonal, RK 21020), and after completion, trans1-T1 competent cells (whole-gold organism, CD 501) were transformed, and the single clone was picked for PCR verification and the positive clone was subjected to sequencing verification. The plasmid sequenced correctly was extracted and named pKSAB4.
The nucleotide sequence of pKSAB.sup.4 vector is sequence 8, wherein the 1 st to 3282 nd and 5017 th to 6152 th of sequence 8 are pSET4s vector skeleton, the 4705 th to 5016 th are P23 promoter, and the 3283 th to 4704 th are sacB gene.
2. Construction of pKSAB4-2025-Luc-2026 knockout plasmid
The upstream homology arm 2025 (SEQ ID NO: 4) and downstream homology arm 2026 (SEQ ID NO: 5) were amplified using 2025-F/2025-R, 2026-F/2026-R, respectively, as primers and Streptococcus agalactiae 2603V/R (ATCC, BAA-611) as templates;
using CP25-F/CP25-R as a primer, synthesizing a CP25 promoter sequence as a template, amplifying the CP25 promoter (sequence 6);
amplifying the Luc gene (sequence 7) by using Luc-F/Luc-R as a primer and pGL3-basic plasmid as a template;
And (3) performing overlap PCR on the CP25 promoter and Luc by using the CP25-F/Luc-R as a primer to obtain the CP25-Luc.
As a result of the above PCR products being verified by agarose gel electrophoresis, the upstream homology arm 2025 had a size of 775bp, the downstream homology arm 2026 had a size of 775bp, and the CP25-Luc overlap PCR product had a size of 1790bp, as shown in FIG. 2.
PKSAB of the above-prepared pKSAB of 1 was double digested with BamHI/HindIII at 37℃for 2 hours to give pKSAB after digestion.
And (3) recovering the PCR products and the digestion products by using a DNA product recovery kit, performing seamless cloning on the upstream homology arm 2025, the downstream homology arm 2026, the CP25-Luc and the digestion pKSAB by using 2X MultiF Seamless Assembly Mix and incubating at 50 degrees for 50-min, transforming the Trans1-T1 competent cells after completion, and selecting a monoclonal and using 2025-F/2026-R as primers for PCR verification.
As a result, as shown in FIG. 3, lanes 1-2 are all amplification products, and as a result, the PCR product size was 3380bp, which was consistent with the target size, and sequencing was performed.
The plasmid sequenced correctly was extracted and named pKSAB-2025-Luc-2026.
Plasmid pKSAB-2025-Luc-2026 is a plasmid obtained by inserting a fragment containing the Luc gene expression cassette between BamHI/HindIII cleavage sites of pKSAB vector.
The fragment containing the Luc gene expression cassette is a fragment comprising the upstream homology arm 2025, the CP25 promoter, the Luc gene and the downstream homology arm 2026 in sequence, and the nucleotide sequence thereof is a fragment comprising the upstream homology arm 2025 shown in sequence 4, the CP25 promoter shown in sequence 6, the Luc gene shown in sequence 7 and the downstream homology arm 2026 shown in sequence 5 in sequence.
3. Preparation of Streptococcus agalactiae competence
(1) Inoculating streptococcus agalactiae 2603V/R into 5mL of M9 culture medium containing 1% (mass percent g: mL) tyrosine and 0.3% yeast extract, and standing and culturing for 10h in a constant temperature incubator with 5% CO2 at 37 ℃;
(2) Adding the cultured streptococcus agalactiae into 50ml of M9 culture medium containing 1% tyrosine, 0.3% glycine and 0.3% yeast extract, and standing overnight at 37deg.C in a constant temperature incubator with 5% CO 2;
(3) Adding the cultured streptococcus agalactiae into 500ml of the same preheated culture medium at 37 ℃, and carrying out stationary culture for 1h in a constant-temperature incubator with 5% CO 2 at 37 ℃;
(4) Centrifuging at 10000r/min for 10min, and removing supernatant;
(5) Washing with 150ml of precooled solution containing 25% PGE2000 and 10% glycerol once, centrifuging at 4deg.C and 10000r/min for 10min, and removing supernatant;
(6) Re-suspending with 1mL pre-cooled 25% PGE2000 and 10% glycerol solution, and packaging into 1.5mL sterile Ep tubes, 50 μl each, and storing at-80deg.C to obtain Streptococcus agalactiae competent cells.
4. Construction of Streptococcus agalactiae luminous bacteria
1) Electric rotating device
Taking the streptococcus agalactiae competent cells prepared in advance in the 3, naturally melting the competent cells, adding 1 mug of the plasmid pKSAB4-2025-Luc-2026 prepared in the 2 as electrotransformation DNA, carefully blowing and mixing uniformly, and standing on ice for 5min; carefully adding competent cells into a precooled 0.1cm electric shock cup, and noticing that no bubbles can be generated in the adding process; placing the electric shock cup into an electrotransformation instrument for electrotransformation under the conditions of 1.25kV,200 omega and 25 mu F; immediately adding 1mL of preheated resuscitation fluid after the electric transfer is finished; shake culturing in a shake incubator at 28deg.C for 3 hr; after the culture is finished, centrifuging for 5min at normal temperature of 8000r/min, removing supernatant, and adding 100 mu L of sterile PBS to resuspend thalli; the resuspended cells were spread evenly on THB solid plates containing spectinomycin and incubated upside down at 28 ℃.
2) Identification of homologous recombination
After single colony is grown, selecting a single clone to 5mL of fresh THB liquid culture medium containing spectinomycin, transferring the single clone to the THB liquid culture medium at 28 ℃ for 3 times continuously, transferring the single clone by 10%, placing the single clone in a 37 ℃ incubator for culture, streaking the single clone on a spectinomycin-resistant THB plate after the single clone grows to a plateau, and placing the plate in the 37 ℃ incubator for culture. Colony PCR was performed on the single clone grown on the plate using ID-F/CP25-R as primer, and clones exchanged on the upstream homology arm were screened (1095 bp target fragment was obtained).
As shown in FIG. 4, the size of the PCR product is identical to the target size 1095bp, and the exchange is realized, so that the strain with the upstream homology arm exchange is obtained.
3) Identification of Streptococcus agalactiae light-emitting bacteria
Clones identified in 2) above were transferred to 5mL of fresh non-anti-THB liquid medium, serially passaged 3 times at 28℃and cultured in a 37℃incubator after 10% transfer, after growing to the plateau phase, bacterial liquid was diluted with PBS and spread on THB solid plates without anti-containing 0.75M sucrose (clones lost by sacB gene reverse screening, obtained PKSAB4 plasmids), and the plates were placed in a 37℃incubator for culture.
After the monoclonal colony is grown, using ID-F/ID-R as a primer to verify cloning, wherein the PCR product is 1802bp of wild streptococcus agalactiae, and the PCR product is 3592bp of luminous streptococcus agalactiae, namely the luminous streptococcus agalactiae.
As a result, as shown in FIG. 5, it was found that 3592bp of Streptococcus agalactiae was obtained.
Streptococcus agalactiae light-emitting bacteria are obtained by inserting a fragment consisting of a CP25 promoter and a Luc gene (the nucleotide sequence consists of sequence 6 and sequence 7) between SAG2025 and SAG2026 genes of Streptococcus agalactiae 2603V/R (GenBank: AE009948, 31-JAN-2014), at the 2003475 genome.
2. Verification of Streptococcus agalactiae light-emitting bacteria
1. Verification of strains
The wild Streptococcus agalactiae (designated as wild strain in the figure) and Streptococcus agalactiae light-emitting bacteria (designated as 1-7 in the figure) obtained above were transferred to a non-anti-THB liquid medium, respectively, and after growing to the plateau, 50. Mu.L of each of the wild Streptococcus agalactiae culture solution and Streptococcus agalactiae light-emitting bacteria culture solution was aspirated and added to a detection plate, and 50. Mu.L of Bright-Glo ™ luciferase detection reagent (Promega, E2610) was added, respectively, and the luminescence intensity of the bacteria was detected using a GLOMAX luminescence detector.
As a result, as shown in FIG. 6, the luminous intensity of the constructed Streptococcus agalactiae luminous bacteria was improved by hundreds to thousands times compared with that of the wild Streptococcus agalactiae, which indicates that the construction of Streptococcus agalactiae luminous bacteria based on the luciferase reporter system was successful.
2. Real-time observation in animal body
The streptococcus agalactiae luminous bacteria No. 3 in the above 1 was randomly selected, cultured until the OD600 = 0.2, centrifuged at 8000 Xg for 3 minutes, the strain was collected, washed once with PBS and resuspended in PBS, and C57BL/6 mice 1X 10 8 CFU/mouse were intraperitoneally injected with fluorescein (D-potassium luciferin, mikrin, D812647) at 150 mg/kg of animal body weight (or 10. Mu.L/g of a stock solution of fluorescein dissolved with DPBS) 10-15 minutes after 5 hours post-infection, and the luminous status of the bacteria in the mice was photographed using a small animal imager.
As a result, as shown in FIG. 7, it was found that a very clear luminescent region was present in the abdomen of two mice, indicating that luciferase could be expressed in Streptococcus agalactiae and that tracing of the agalactiae could be achieved in the mice.
Claims (10)
1. A Streptococcus agalactiae luminous bacterium is a strain obtained by tracelessly inserting a fragment consisting of a target luminous gene promoter and a target luminous gene into a Streptococcus agalactiae genome.
2. A method of preparing the streptococcus agalactiae luminous bacteria of claim 1, comprising the steps of: the recombinant plasmid is led into streptococcus agalactiae, and then homologous recombination and reverse screening marker sacB identification are carried out to obtain streptococcus agalactiae luminous bacteria;
The recombinant plasmid is obtained by inserting a fragment containing a target luminous gene promoter and a target luminous gene into a shuttle plasmid;
The shuttle plasmid comprises a pSET4s vector skeleton, sacB genes and a promoter for driving the expression of the sacB genes;
the nucleotide sequence of the pSET4s vector framework is a sequence 3;
the nucleotide sequence of the sacB gene is sequence 2.
3. The method according to claim 2, characterized in that:
The promoter for driving sacB gene expression is a P23 promoter, and the nucleotide sequence of the promoter is a sequence 1;
The nucleotide sequence of the shuttle plasmid is sequence 8.
4. A method according to claim 2 or 3, characterized in that:
The fragment containing the target light emitting gene promoter and the target light emitting gene includes an upstream homology arm for homologous recombination into the streptococcus agalactiae genome, the target light emitting gene promoter, the target light emitting gene and the downstream homology arm for homologous recombination into the streptococcus agalactiae genome.
5. The method according to claim 4, wherein:
The target luminous gene promoter is a CP25 promoter;
The target luminescent gene is Luc gene.
6. Streptococcus agalactiae light-emitting bacteria prepared by the method of any one of claims 2-5.
7. Use of the streptococcus agalactiae light-emitting bacterium of claim 1 or 6 or the method of any one of claims 2-5 in the preparation of a streptococcus agalactiae infected cell model or an animal model of streptococcus agalactiae infection;
Or Streptococcus agalactiae light-emitting bacteria according to claim 1 or 6 or the use of a method according to any one of claims 2 to 5 for the preparation of a product for in vivo observation of Streptococcus agalactiae by tracing in an animal.
8. A streptococcus agalactiae tracer observation product for use in an animal comprising the luminescent streptococcus agalactiae of claim 1 or 6.
9. The shuttle plasmid or the recombinant plasmid of any one of claims 2-5.
10. The use of the shuttle plasmid of claim 9 or the recombinant plasmid in any of the following:
1) Preparing a streptococcus agalactiae infection cell model or an animal model of streptococcus agalactiae infection;
2) The streptococcus agalactiae tracing and observing product in animals is prepared.
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