CN114540351B - sRNA of targeted klebsiella pneumoniae MdtABC efflux pump and application thereof in preparation of tetracycline antibiotic resistant strain - Google Patents
sRNA of targeted klebsiella pneumoniae MdtABC efflux pump and application thereof in preparation of tetracycline antibiotic resistant strain Download PDFInfo
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Abstract
The invention discloses sRNA of a targeted klebsiella pneumoniae MdtABC efflux pump and application thereof in preparation of tetracycline antibiotic resistant strains. The sRNA-120 sRNA of the negative regulation MdtABC efflux pump is obtained through the bioinformatics analysis of the klebsiella pneumoniae tigecycline-induced drug-resistant strain. The klebsiella pneumoniae clinically sensitive strain KPS1013 is transformed into a 120sRNA knockout strain KPS-delta 120sRNA. The change in susceptibility of klebsiella pneumoniae to antibiotics before and after 120sRNA knockout was detected by Minimum Inhibitory Concentration (MIC) assay. As a result, it was found that the strain after 120sRNA knockout had 4 times higher resistance to tigecycline, omacycline, and epothilone than the strain before knockout. The result reveals that the 120sRNA has reverse regulation and control effect on the expression of mdtABC efflux pump genes, and the drug resistance of klebsiella pneumoniae can be improved after the 120sRNA is knocked out. The invention provides a new technical means for researching sRNA to participate in regulation and control of bacterial drug resistance mechanism and research of anti-Klebsiella pneumoniae drug resistance bacteria drugs.
Description
Technical Field
The invention relates to a small RNA and application thereof, in particular to a small RNA targeting a klebsiella pneumoniae MdtABC efflux pump and application thereof in preparation of a tetracycline antibiotic klebsiella pneumoniae drug-resistant strain. The invention belongs to the technical field of medicines.
Background
Klebsiella pneumoniae (Klebsiella pneumoniae) is a gram-negative bacterium of the genus Enterobacter, klebsiella. In the natural world and in healthy humans, when the immunity of the body is reduced or the health is problematic, various inflammations such as pneumonia, meningitis, urinary tract infection, blood infection, liver abscess and other invasive infections can be caused.
Mdabc efflux pumps belong to the five major efflux pump families related to drug resistance in gram negative bacteria, the drug resistant nodular cell division family (Resistance Nodulation-cell Division superfamily, RND), which exist in the bacterial inner and outer membrane interstices, are associated with the pumping of a variety of antibiotics, harmful endogenous metabolites and toxic exogenous compounds, which are generally highly expressed in klebsiella pneumoniae tigecycline-resistant strains and are regulated by a variety of regulatory factors.
sRNA (small RNA), a non-coding small RNA with the size of 50-500 nt. At present, various researches show that sRNA participates in the expression regulation of various genes in bacteria, and plays an important role in bacterial virulence, amino acid metabolism, drug resistance and the like. Which further regulates the expression of the corresponding target gene, mainly by binding to the gene. In general, the large number of sRNAs involved in regulating the same gene makes the research on specific sRNAs cumbersome and difficult, and the related research is not clear and comprehensive.
The invention identifies sRNA of a specific targeting mdtABC efflux pump gene and finds that the sRNA has reverse regulation effect on the expression of the mdtABC gene. The regulatory effect of sRNA on mdtABC affects the sensitivity of Klebsiella pneumoniae to tigecycline (omadacyline) and two novel tetracycline antibiotics, omadacyline and eladacyline. Provides a new technical means for construction of drug-resistant bacteria, clinical treatment and drug research and development of drug-resistant bacteria.
Disclosure of Invention
One of the purposes of the invention is to provide sRNA of a targeted Klebsiella pneumoniae MdtABC efflux pump.
The second purpose of the invention is to provide the application of the sRNA in preparing the Klebsiella pneumoniae tetracycline antibiotic resistant strain.
The invention further aims to provide a method for preparing the Klebsiella pneumoniae tetracycline antibiotic resistant strain, the Klebsiella pneumoniae tetracycline antibiotic resistant strain prepared by the method and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical means:
the invention screens sRNA-120 sRNA of negative regulation mdtABC efflux pump genes through bioinformatics analysis of klebsiella pneumoniae tigecycline-induced drug-resistant strains, builds 120sRNA knockout plasmid pK18 mcbssacB-delta 120CmR by using a molecular cloning and homologous recombination method, and changes clinical klebsiella pneumoniae sensitive strain KPS into 120sRNA knockout strain KPS-delta 120sRNA. The change of the expression level of mdtABC gene before and after the knocking-out of 120sRNA is detected by a real-time quantitative PCR (qPCR) method and a Minimum Inhibitory Concentration (MIC) method, and the sensitivity of Klebsiella pneumoniae to tigecycline (tigecycline), omaacyline (omadacycine) and elanocycine (eravacycine) antibiotics is changed. The qPCR experimental result shows that after the 120sRNA is knocked out, the expression quantity of the 120sRNA is reduced, and the expression quantity of mdtA and mdtC is increased; and the strain after knockout was 4-fold resistant to tigecycline (tigecycline), omacycline (omadacycine) and epothilone (eradacycine) before knockout (based on MIC values).
Therefore, on the basis of the research, the invention firstly provides sRNA for targeting the Klebsiella pneumoniae MdtABC efflux pump, wherein the sRNA is named as 120sRNA and is obtained by transcribing a DNA sequence shown in SEQ ID NO. 1.
Furthermore, the invention also provides application of the sRNA of the targeted Klebsiella pneumoniae MdtABC efflux pump in preparation of Klebsiella pneumoniae tetracycline antibiotic resistant strains.
Preferably, the tetracycline antibiotics include tigecycline (tigecycline), omacycline (omadacycine), and elanocycline (eravacycine).
Still further, the invention also provides a method for preparing the klebsiella pneumoniae tetracycline antibiotic resistant strain, which comprises the step of knocking out 120sRNA of the klebsiella pneumoniae sensitive strain.
Preferably, the method specifically comprises the following steps:
(1) Preparation of 120sRNA knockout plasmid pK18 mcbsaccB-Delta120 CmR
1) Designing 4 pairs of primers related to upstream and downstream homology arms of sRNA through a primer-blast function in NCBI, wherein the primer pairs 120U1-F/R and 120D1-F/R are respectively used for amplifying the upstream and downstream homology arms of the sRNA; the primer pairs 120U2-F/R and 120D2-F/R are respectively amplified to obtain sRNA upstream and downstream homology arms with enzyme cutting sites and for overlap PCR;
upstream and downstream homology arm primers:
120U1-F：GGTAAGGTGGCCCAGGAG
120U1-R：GTGAGAAACAACTATTTTGCACAGC
120D1-F：GGGTCGGGATGCCAGTATTC
120D1-R：TCGGCAATCTGACTACACTGG
primers with cleavage sites:
120U2-F：
120U2-R：
GAATACTGGCATCCCGACCCGTGAGAAACAACTATTTTGCACAGCG
120D2-F：
GCAAAATAGTTGTTTCTCACGGGTCGGGATGCC
120D2-R：
2) The genome DNA of Klebsiella pneumoniae sensitive to antibiotics is used as a template, and primer pairs 120U1-F/R and 120D1-F/R are used for amplification to obtain sRNA upstream and downstream homology arms 120U1 and 120D1;
3) The sRNA upstream and downstream homology arms 120U1 and 120D1 are used as templates, and sRNA upstream and downstream homology arms 120U2 and 120D2 with enzyme cutting sites and for overlap PCR are obtained through amplification of primer pairs 120U2-F/R and 120D 2-F/R;
4) Connecting the sRNA upstream and downstream homology arms 120U2 and 120D2 into sRNA upstream and downstream homology arm fusion fragments through overlap PCR;
5) Double digestion of the pK18 mcbsach plasmid and the sRNA upstream and downstream homology arm fusion fragments was performed using HindIII, ecoRI;
6) The fusion fragments of the pK18mcbsACB plasmid and the upstream and downstream homology arms of sRNA after enzyme digestion are connected by T4 DNA ligase;
7) The connection product is transformed into E.coli DH5 alpha competent cells, and positive clones are screened by kanamycin agar plates;
8) The PCR further verifies positive clones, and the pK18 mcbsach-delta 120 plasmid is extracted after the amplification culture;
9) PCR (polymerase chain reaction) with pBAD33 plasmid as a template to obtain a chloramphenicol resistance fragment;
resistance fragment primers:
CmR-F：
CmR-R：
10 After cleavage of the pK18 mcbsach- Δ120 plasmid and chloramphenicol resistance fragment using HindIII, T4 DNA ligase was ligated;
11 Transformation of the ligation products into E.coli DH 5. Alpha. Competent cells, screening positive clones with kanamycin+chloramphenicol agar plates;
12 PCR to further verify positive clones, extracting pK18 mcbsaccB-delta 120CmR plasmid after amplification culture;
(2) Preparation of Klebsiella pneumoniae drug-resistant strain
1) Transforming the extracted plasmid pK18 mcbsaccB-delta 120CmR into antibiotic-sensitive clinical Klebsiella pneumoniae competence, and screening positive clones by kanamycin and chloramphenicol agar plates;
2) And (3) carrying out streak subculture and sucrose screening on positive clones, carrying out PCR verification on the obtained clone strain, and carrying out sequencing confirmation on a correct PCR product to obtain the Klebsiella pneumoniae with the knocked-out 120sRNA, namely the Klebsiella pneumoniae tetracycline antibiotic resistant strain, and storing for later use.
Preferably, the tetracycline antibiotics include tigecycline (tigecycline), omacycline (omadacyline), and elavidin (eradacyline).
Preferably, the antibiotic-sensitive clinical klebsiella pneumoniae is klebsiella pneumoniae KPS, the klebsiella pneumoniae is named KPS1013, the klebsiella pneumoniae is named klebsiella pneumoniae (Klebsiella pneumoniae) and is stored in the general microbiological center of the China Committee for culture Collection of microorganisms, the address is 3 # of national institute of microbiology, national institute of sciences of China, the collection number is CGMCC NO.24290, and the storage date is 2022, 1 month and 11 days.
The Klebsiella pneumoniae tetracycline antibiotic resistant strain prepared by the method is also within the protection scope of the invention.
Preferably, the resistance of the Klebsiella pneumoniae tetracycline antibiotic resistant strain to tigecycline (Tigecycline), omacycline (omadacyline) and eprosacycline (eradacyline) is 4 times of that of the strain before knockout.
Furthermore, the invention also provides application of the klebsiella pneumoniae tetracycline antibiotic resistant strain in research of sRNA participating in regulation of bacterial drug resistance mechanism and development of anti-klebsiella pneumoniae drug resistance.
Compared with the prior art, the invention has the beneficial effects that:
the sRNA-120 sRNA which can be specifically combined with the mdtABC target gene and negatively regulate the mdtABC efflux pump is obtained through bioinformatics analysis of the klebsiella pneumoniae tigecycline-induced drug-resistant strain. The research shows that after 120sRNA knockout, the expression quantity of mdtA and mdtC genes is increased; and the drug resistance of the strain after knockout to tigecycline (tigecycline), omacycline (omadacycine) and epothilone (eradacycine) is 4 times that of the strain before knockout. The result reveals that the 120sRNA has reverse regulation and control effect on the expression of mdtABC efflux pump genes, and the 120sRNA can improve the drug resistance of klebsiella pneumoniae after being knocked out, and can be used for preparing klebsiella pneumoniae tetracycline antibiotic resistant strains. The invention provides a new technical means for researching sRNA to participate in the regulation of a bacterial drug resistance mechanism and the development of anti-Klebsiella pneumoniae drug resistance medicines.
Drawings
FIG. 1 shows the results of amplification electrophoresis of upstream and downstream homology arms of 120 sRNA;
wherein: m is 100bp DNA maker,Up and Down is the upstream homology arm;
FIG. 2 shows the results of PCR detection of 120sRNA gene changes in the genome before and after knocking out of a Klebsiella pneumoniae sensitive strain;
wherein: m is 100bp DNA maker,After which is a fusion fragment of an upstream homology arm and a downstream homology arm of 120sRNA after knockout, and Before is a full-length fragment of the 120sRNA and the upstream homology arm and the downstream homology arm Before knockout;
FIG. 3 shows qPCR detection results of 120sRNA, mdtA and mdtC expression changes before and after the knocking out of the Klebsiella pneumoniae sensitive strain;
FIG. 4 shows MIC changes before and after knockout of Klebsiella pneumoniae sensitive strains.
Detailed Description
The advantages and features of the present invention will become more apparent from the following description of the embodiments. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions in the details and form of the present invention may be made without departing from the spirit and scope of the invention, but that these changes and substitutions fall within the scope of the invention.
EXAMPLE 1 sRNA screening
The invention screens and obtains sRNA-120 sRNA which can be specifically combined with an MdtABC target gene and negatively regulates an MdtABC efflux pump through bioinformatics analysis of a Klebsiella pneumoniae tigecycline-induced drug-resistant strain, wherein the 120sRNA is obtained by transcribing a DNA sequence shown in SEQ ID NO. 1.
Example 2 application of 120sRNA in preparation of Klebsiella pneumoniae drug-resistant strain
1. Preparation of 120sRNA knockout plasmid pK18 mcbsaccB-Delta120 CmR
1) Designing 4 pairs of primers related to upstream and downstream homology arms of sRNA through a primer-blast function in NCBI, wherein the primer pairs 120U1-F/R and 120D1-F/R are respectively used for amplifying the upstream and downstream homology arms of the sRNA; the primer pairs 120U2-F/R and 120D2-F/R are respectively amplified to obtain sRNA upstream and downstream homology arms with enzyme cutting sites and for overlap PCR;
upstream and downstream homology arm primers:
120U1-F：GGTAAGGTGGCCCAGGAG
120U1-R：GTGAGAAACAACTATTTTGCACAGC
120D1-F：GGGTCGGGATGCCAGTATTC
120D1-R：TCGGCAATCTGACTACACTGG
primers with cleavage sites (lower case letters are cleavage site and protecting base):
120U2-F：
120U2-R：
GAATACTGGCATCCCGACCCGTGAGAAACAACTATTTTGCACAGCG
120D2-F：
GCAAAATAGTTGTTTCTCACGGGTCGGGATGCC
120D2-R：
2) The primer pair 120U1-F/R and 120D1-F/R is used for amplifying to obtain sRNA upstream and downstream homology arms (120U 1 and 120D 1) by taking the tigecycline sensitive clinical Klebsiella pneumoniae KPS (CGMCC NO. 24290) genome DNA as a template, and the electrophoresis result is shown in figure 1;
wherein, the extraction of the Klebsiella pneumoniae KPS genome sensitive to tigecycline is carried out according to the following method: genome DNA WAs collected by using Buffer GL, protease K, RNase A, buffer GB and absolute ethanol to lyse cells, washing the collection column with Buffer WA and Buffer WB, and finally using ddH 2 O was dissolved to obtain genomic DNA.
3) Amplifying by using the sRNA upstream and downstream homology arms as templates through primer pairs 120U2-F/R and 120D2-F/R to obtain sRNA upstream and downstream homology arms (120U 2 and 120D 2) with enzyme cutting sites and for overlap PCR;
4) The sRNA upstream and downstream homology arms 120U2 and 120D2 are connected into upstream and downstream homology arm fusion fragments through overlap PCR;
the first step reaction system: 120U 2. Mu.l, 120D 2. Mu.l, dNTP Mix 1. Mu.l, 2X Phanta Max Buffer. Mu.l, ddH 2 O17 μl; the reaction procedure: 94℃for 2min, (94℃for 10s,62℃for 30s,72℃for 1min, 5 cycles total), 72℃for 2min.
And a second reaction system: to the first step system, 2. Mu.l each of 120U2-F and 120D2-R was added, and 1. Mu.l each of Phanta Max Super-Fidelity DNA Polymerase was added; the reaction procedure: 95℃for 3min, (95℃15s,65℃15s,72℃40s, total of 35 cycles), 72℃for 5min.
5) Double digestion of the pK18 mcbsach plasmid and the sRNA upstream and downstream homology arm fusion fragments was performed using HindIII, ecoRI;
6) The fusion fragments of the pK18mcbsACB plasmid and the upstream and downstream homology arms of sRNA after enzyme digestion are connected by T4 DNA ligase;
the T4 ligation system was 3. Mu.l of vector cleavage product, 7. Mu.l of insert cleavage product, 10. Mu.l of Enzyme Solution, and the reaction procedure was 30min at 16 ℃;
7) The connection product is transformed into E.coli DH5 alpha competent cells, and positive clones are screened by kanamycin agar plates;
the conversion method is a heat shock conversion method, and comprises the following specific operations: thawing chemically competent cells on ice, adding 10. Mu.l of DNA mixture (9. Mu.l of ligation product, transformation Enhancer. Mu.l) to 100. Mu.l of competent cells, standing on ice for 30min, standing on ice for 90s at 42℃for 3min, adding 890. Mu.l of LB broth medium, and shake culturing at 37℃and 200rpm for 1h; 200. Mu.l of the cultured bacterial liquid was uniformly spread on LB agar plates containing kanamycin, and cultured at 37℃for 16 to 18 hours.
8) The PCR further verifies positive clones, and the pK18 mcbsach-delta 120 plasmid is extracted after the amplification culture;
9) PCR (polymerase chain reaction) with pBAD33 plasmid as a template to obtain a chloramphenicol resistance fragment;
resistance fragment primer (lower case part is enzyme cleavage site and protecting base):
CmR-F：
CmR-R：
10 After cleavage of the pK18 mcbsach- Δ120 plasmid and chloramphenicol resistance fragment using HindIII, T4 DNA ligase was ligated;
11 Transformation of the ligation products into E.coli DH 5. Alpha. Competent cells, screening positive clones with kanamycin+chloramphenicol agar plates;
12 PCR was further confirmed for positive clones, and pK18 mcbsaccB-Delta120 CmR plasmid was extracted after expansion culture.
2. Preparation of drug-resistant strains
1) Transforming the extracted plasmid pK18 mcbsacoB-delta 120CmR into the clinical Klebsiella pneumophila KPS competence sensitive to tigecycline, and screening positive clones by using a kanamycin and chloramphenicol agar plate;
2) After positive cloning is subjected to streak subculture and sucrose screening, the obtained clone strain is subjected to PCR verification, and after correct PCR product sequencing confirmation, the Klebsiella pneumoniae with 120sRNA knocked out is obtained, and the clone strain is named as KPS-delta 120sRNA and is preserved.
3. sRNA gene variation in genome before and after knocking out of Klebsiella pneumoniae sensitive strain
The change in the expression level of mdabc gene before and after 120sRNA knockout was detected by real-time quantitative PCR (qPCR).
RNA extraction: the cells were lysed using TransZol Up, RNA was precipitated by adding chloroform and isopropanol, and finally washed with 75% ethanol and air-dried, the RNA was dissolved in RNA-dissolving solution, and the concentration was detected and inverted to cDNA.
qRT-PCR: the system per hole is as follows: cDNA (50 ng/. Mu.l) 2. Mu.l, primer-F0.8. Mu.l, primer-R0.8. Mu.l, ddH2O 6.4. Mu.l, TB Green Premix Ex Taq II. Mu.l; the reaction procedure: 95℃for 30s, (95℃for 5s,60℃for 34s, 40 cycles total), 95℃for 15s,60℃for 1min,95℃for 15s; each sample was set up with 4 replicates, passing 2 -△△CT The method calculates the difference in target gene expression between samples.
Results:
FIG. 2 shows sRNA gene changes in the genome of Klebsiella pneumoniae susceptible strains before and after knockout; FIG. 3 shows qPCR changes before and after knockout of Klebsiella pulmonary infection sensitive strains. The qPCR experimental result shows that after the 120sRNA is knocked out, the expression level of the 120sRNA is reduced, and the expression levels of mdtA and mdtC are increased.
4. Minimum Inhibitory Concentration (MIC) detection of sRNA knockdown strains
After 2-fold gradient dilution of 512. Mu.g/ml of antibiotic mother liquor (tigecycline, omadacycline or eravacyline), the mixture was diluted with a bacterial solution (1.5X10 6 CFU/ml) according to 1:1, wherein the final concentration of each row of antibiotics is 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 mug/ml, and the final concentration of each hole bacterial liquid is 7.25X10 5 CFU/ml. Incubation was performed at 37℃for 24h, and changes in antibiotic susceptibility were recorded for the different samples.
Results:
fig. 4 shows MIC changes before and after knocking out 120sRNA in a klebsiella pneumoniae clinically sensitive strain, and the experimental result shows that the strain after knocking out 120sRNA has 4 times of drug resistance to Tigecycline (TGC), omaacycline (omadacyline, oma) and epothilone (Era) than the strain before knocking out.
The above results revealed that 120sRNA has a reverse regulatory effect on the expression of mdtABC efflux pump gene. The invention provides a new technical means for researching sRNA to participate in regulation and control of bacterial drug resistance mechanism and research of anti-Klebsiella pneumoniae drug resistance drugs.
Sequence listing
<110> university of Dalian medical science
<120> sRNA of targeted Klebsiella pneumoniae MdtABC efflux pump and application thereof in preparation of tetracycline antibiotic resistant strain
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 374
<212> DNA
<213> Klebsiella pneumoniae
<400> 1
cccctcacaacggaatgtgaaatgctaaaattaacccctggcgcgaataaagcgaatgcgattctgatccgtattgtcttgtttcttatcggcggtatcgccatcatcaccggaatgtgcctcttcagcggtgatatgtggatgctcaccccgcgggcggaacagtatcagcccgccaccggcgatctggtggatgtcgtcatctatgaaccgagtaaatttttcttcgccctgctctacgtgtttattattaccctggtctggctgctggcggtacgtaaaatcccctggcggccgatacagcatcctatattagcgctgataatactgattgccgccgggatatttattttcctccacccggaggtcacccc
Claims (8)
1. The sRNA for targeting the Klebsiella pneumoniae MdtABC efflux pump is characterized in that the sRNA is named as 120sRNA and is obtained by transcription of a DNA sequence shown in SEQ ID NO. 1.
2. The use of the sRNA of the targeted klebsiella pneumoniae MdtABC efflux pump of claim 1 for preparing a klebsiella pneumoniae tetracycline antibiotic resistant strain.
3. The use of claim 2, wherein the tetracycline antibiotic comprises tigecycline (tigecycline), omacycline (omadacyline), and elaxacine (eradacyline).
4. The method for preparing the klebsiella pneumoniae tetracycline antibiotic resistant strain is characterized by comprising the step of knocking out 120sRNA of a klebsiella pneumoniae sensitive strain, wherein the 120sRNA is obtained by transcribing a DNA sequence shown in SEQ ID NO. 1.
5. The method of claim 4, comprising the steps of:
(1) Preparation of 120sRNA knockout plasmid pK18 mcbsaccB-Delta120 CmR
1) Designing 4 pairs of primers related to upstream and downstream homology arms of sRNA through a primer-blast function in NCBI, wherein the primer pairs 120U1-F/R and 120D1-F/R are respectively used for amplifying the upstream and downstream homology arms of the sRNA; the primer pairs 120U2-F/R and 120D2-F/R are respectively amplified to obtain sRNA upstream and downstream homology arms with enzyme cutting sites and for overlap PCR;
upstream and downstream homology arm primers:
120U1-F：GGTAAGGTGGCCCAGGAG
120U1-R：GTGAGAAACAACTATTTTGCACAGC
120D1-F：GGGTCGGGATGCCAGTATTC
120D1-R：TCGGCAATCTGACTACACTGG
primers with cleavage sites:
120U2-F：
GATAGCTACAGACTTAGCtattcccaagcttGGTAAGGTGGCCCAGGAG
120U2-R：GAATACTGGCATCCCGACCCGTGAGAAACAACTATTTTGCACAGCG
120D2-F：
GCAAAATAGTTGTTTCTCACGGGTCGGGATGCC
120D2-R：TCAGCTGTCAGATGCgtaaccggaattcTCGGCAATCTGACTACACTGG
2) The genome DNA of clinical klebsiella pneumoniae with antibiotic sensitivity is used as a template, and the primer pairs 120U1-F/R and 120D1-F/R are used for amplification to obtain sRNA upstream and downstream homology arms 120U1 and 120D1;
3) The sRNA upstream and downstream homology arms 120U1 and 120D1 are used as templates, and sRNA upstream and downstream homology arms 120U2 and 120D2 with enzyme cutting sites and for overlap PCR are obtained through amplification of primer pairs 120U2-F/R and 120D 2-F/R;
4) Connecting the sRNA upstream and downstream homology arms 120U2 and 120D2 into sRNA upstream and downstream homology arm fusion fragments through overlap PCR;
5) Double digestion of the pK18 mcbsach plasmid and the sRNA upstream and downstream homology arm fusion fragments was performed using HindIII, ecoRI;
6) The fusion fragments of the pK18mcbsACB plasmid and the upstream and downstream homology arms of sRNA after enzyme digestion are connected by T4 DNA ligase;
7) The connection product is transformed into E.coli DH5 alpha competent cells, and positive clones are screened by kanamycin agar plates;
8) The PCR further verifies positive clones, and the pK18 mcbsach-delta 120 plasmid is extracted after bacterial culture;
9) PCR (polymerase chain reaction) with pBAD33 plasmid as a template to obtain a chloramphenicol resistance fragment;
resistance fragment primers:
CmR-F：
TAGATGACTACTATCAcccaagcttTTACGCCCCGCCCTG
CmR-R：CGAGGCTCGGACTGTCCAGGcccaagcttATGGAGAAAAAAATCACTGG
10 After cleavage of the pK18 mcbsach- Δ120 plasmid and chloramphenicol resistance fragment using HindIII, T4 DNA ligase was ligated;
11 Transformation of the ligation products into E.coli DH 5. Alpha. Competent cells, screening positive clones with kanamycin+chloramphenicol agar plates;
12 PCR to further verify positive clones, extracting pK18 mcbsach-delta 120CmR plasmid after bacterial culture;
(2) Preparation of Klebsiella pneumoniae drug-resistant strain
1) Transforming the extracted plasmid pK18 mcbsaccB-delta 120CmR into antibiotic-sensitive clinical Klebsiella pneumoniae competence, and screening positive clones by kanamycin and chloramphenicol agar plates;
2) And (3) carrying out streak subculture and sucrose screening on positive clones, carrying out PCR verification on the obtained clone strain, and carrying out sequencing confirmation on a correct PCR product to obtain the Klebsiella pneumoniae with the knocked-out 120sRNA, namely the Klebsiella pneumoniae tetracycline antibiotic resistant strain, and storing for later use.
6. The method of claim 5, wherein the tetracycline antibiotic comprises tigecycline (tigecycline), omacycline (omadacycine), and elaxacycline (eradacycine).
7. The method of claim 5, wherein the antibiotic-sensitive clinical klebsiella pneumoniae is klebsiella pneumoniae KPS, which is deposited with the China general microbiological culture collection center with the collection number of CGMCC No.24290 and the date of deposit of 2022, 1 month and 11 days.
8. A klebsiella pneumoniae tetracycline antibiotic-resistant strain prepared according to the method of any one of claims 5-7.
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