CN113416740A - Novel ykkC resistance gene of chloramphenicol and use thereof - Google Patents
Novel ykkC resistance gene of chloramphenicol and use thereof Download PDFInfo
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- CN113416740A CN113416740A CN202110789587.9A CN202110789587A CN113416740A CN 113416740 A CN113416740 A CN 113416740A CN 202110789587 A CN202110789587 A CN 202110789587A CN 113416740 A CN113416740 A CN 113416740A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 43
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 title claims abstract description 40
- 229960005091 chloramphenicol Drugs 0.000 title claims abstract description 40
- 101150016844 gdnC gene Proteins 0.000 title claims abstract description 21
- 206010059866 Drug resistance Diseases 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 claims description 2
- 230000014509 gene expression Effects 0.000 claims 1
- 239000003112 inhibitor Substances 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 4
- 230000001502 supplementing effect Effects 0.000 abstract description 3
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 2
- 230000008261 resistance mechanism Effects 0.000 abstract description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 12
- 101000685990 Homo sapiens Specifically androgen-regulated gene protein Proteins 0.000 description 4
- 102100023355 Specifically androgen-regulated gene protein Human genes 0.000 description 4
- 230000003115 biocidal effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 241000590020 Achromobacter Species 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- 229940122498 Gene expression inhibitor Drugs 0.000 description 1
- 238000007476 Maximum Likelihood Methods 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention relates to a resistance gene of chloramphenicol and application thereof, and particularly discloses a novel resistance gene ykkC _ like of chloramphenicol and application thereof, wherein the ykkC _ like gene sequence is shown as SEQ ID No. 1. Through sequence similarity alignment, no known resistance gene is found on the genome. The invention also discloses a detection method of the gene. The invention provides help for understanding the drug resistance mechanism of chloramphenicol and supplementing a drug resistance database, provides theoretical support for subsequent chloramphenicol resistance, provides a target for solving chloramphenicol resistance, and lays a foundation for research on antibacterial resistance.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a resistance gene of chloramphenicol and application thereof.
Background
The chloramphenicol antibiotic is a broad-spectrum antibiotic separated and extracted from streptomyces, and has good inhibitory effect on a plurality of gram-positive bacteria, gram-negative bacteria and the like. The action mechanism is mainly that the action of the peptide-transferase is blocked by combining with the 50S subunit of the ribosome, thereby influencing the extension of a peptide chain and inhibiting the formation of protein.
Hidden Markov Models (HMM) based on probability can determine implicit parameters of the process from observable parameters, are often used for supplementing gene annotation and gene prediction in the field of research of Antibiotic Resistance Genes (ARGs), and have potential for developing potential novel genes. Traditional gene annotation is usually set by means of a threshold value of sequence similarity, but the method limits the discovery of novel genes and has certain limitation on the annotation of ARGs. The HMM model-based database widely applied at home and abroad comprises Pfam, TIGERFAM, Resfam and the like, and plays a vital role in comprehensively analyzing and mining the ARGs.
The ARGs-OAP is an online ARGs analysis platform, can realize the rapid annotation and quantification of the ARGs of metagenome data, and the database (Structured biological Resistance genes, SARG) contained in the ARGs-OAP integrates two large ARGs databases, namely ARDB and CARD, and lays a solid foundation for the comprehensive analysis of the ARGs by classifying and annotating the ARGs sequences step by step. On the basis of an SARG database, the platform establishes a database SARGfam based on model comparison by applying an HMM model construction method, and can be used for developing potential novel resistance genes.
Disclosure of Invention
Against the background described above, the present invention aims to provide a chloramphenicol resistance gene and its use. The invention discovers a novel resistance gene ykkC _ like of chloramphenicol from a strain of Achromobacter sp, supplements a chloramphenicol resistance database, and lays a solid foundation for deep research of bacterial resistance.
In order to achieve the purpose, the invention adopts the technical scheme that:
the first aspect of the invention provides a chloramphenicol resistance gene, the gene sequence of which is shown in SEQ ID No.1 and is named ykkC _ like gene.
The second aspect of the invention provides the application of the ykkC _ like gene expression inhibitor in preparing a product for reducing the resistance of a subject to chloramphenicol.
The third aspect of the invention is the application of the reagent for detecting the SEQ ID No.1 sequence in the preparation of a kit for detecting the drug resistance of chloramphenicol, and further, the reagent for detecting the SEQ ID No.1 sequence is constructed by qualitatively or quantitatively detecting the SEQ ID No.1 sequence by a PCR method.
The fourth aspect of the invention is to provide the application of ykkC _ like gene as target gene in improving the sensitivity of the subject to chloramphenicol.
The fifth aspect of the invention is to provide a method for detecting chloramphenicol resistance, and further, chloramphenicol resistance is detected based on the sequence information of SEQ ID No.1, which is not for diagnostic purposes.
In the technical scheme of the invention, the chloramphenicol resistance refers to the resistance of gram-positive bacteria and gram-negative bacteria to chloramphenicol.
The technical scheme has the following advantages or beneficial effects:
the invention provides a novel resistance gene of chloramphenicol and application thereof, wherein the novel resistance gene has a sequence shown in SEQ ID No.1 and is named as ykkC _ like gene. Through sequence similarity alignment, no known resistance gene is found on the genome. The invention provides help for understanding the drug resistance mechanism of chloramphenicol and supplementing a drug resistance database, provides theoretical support for subsequent chloramphenicol resistance, provides a target for solving chloramphenicol resistance, and lays a foundation for research on antibacterial resistance.
Drawings
FIG. 1 is a phylogenetic tree diagram between the ykC _ like gene and a known reference gene sequence in example 1, for describing the degree of difference between the ykC _ like gene sequence and the known reference sequence.
Detailed Description
The following examples are only a part of the present invention, and not all of them. Thus, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the invention without making creative efforts, belong to the protection scope of the invention.
Example 1:
1. prediction of Open Reading Frame (ORF) of genome
And (3) applying prodigal software to perform ORF prediction on the pure bacterial genome obtained by subpackaging to obtain an ORF file of the genome.
2. HMM-based model alignment
And (3) comparing the ORF file obtained in the step (1) with a SARGfam database by using a hmmmscan function in HMMER software (parameter setting: -cut _ ga-tblout) to obtain an ORF annotation result based on model comparison. The alignment results are shown in Table 1.
TABLE 1
| Target name | Query id | E-value | sore | Bias |
| ykkC_train_msa | CLB4_1_2097 | 3.8e-22 | 72.3 | 12.8 |
3. Sequence similarity based alignment
And (3) comparing the ORF file obtained in the step (1) with a SARG database by using blastp (parameter setting: evalue 1e-10-max _ target _ seqs 1), and obtaining an ORF annotation result based on sequence similarity. The alignment results are shown in Table 2.
TABLE 2
| Query id | identity | coverage | Annotation |
| CLB4_1_2097 | 42.16 | 0.96 | ykkC |
4. Identification of potentially novel resistance genes
(ii) defining as true the annotation result for the ORF with sequence similarity higher than 70% and coverage higher than 65% in the result of step 3, i.e. known ARGs, whereas an ORF not meeting the threshold is defined as a potential novel gene if there is an annotation result in step 2; if an ORF is not annotated as ARG after alignment based on sequence similarity, but is annotated as ARG in the alignment results of HMM model, the ORF is also considered as a potential novel gene. Through screening, the pure bacterium genome is found to have a potential resistance gene ykkC _ like of the novel chloramphenicol. The reference sequence corresponding to the ykkC gene in the SARG database and the gene sequence of ykkC _ like are subjected to multiple sequence alignment by using ClustalW, a maximum likelihood phylogenetic tree is constructed by using MEGA, the topological structure of the tree is evaluated 1000 times by using a bootstrap method, and the difference degree between the potential novel gene ykkC _ like and the known reference gene sequence is further determined (as shown in fig. 1).
Example 2
The strain containing the ykkC _ like gene is purified and separated, and the MIC value of the strain to the minimum inhibitory concentration of chloramphenicol is detected, and then the MIC value of the strain to the chloramphenicol is up to 120mg/L, which indicates that the ykkC _ like gene enables the strain to have resistance to the chloramphenicol, and the ykkC _ like is a drug-resistant gene of the chloramphenicol.
The above description is only for the preferred embodiment of the present invention and is not intended to limit the scope of the present invention, and all equivalent modifications made by the contents of the present specification and the drawings, or applied directly or indirectly to other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> novel ykkC resistance gene of chloramphenicol and use thereof
<130> CP121010589C
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 318
<212> DNA
<213> Achromobacter
<400> 1
atgacctgga tcatcctggt tctcgcaggc ctgttcgaaa tcgtctgggc cgtaggcctg 60
aaatacaccc acggattcac ccgcttctgg ccatcggcga tcaccgccgg cggcatggcc 120
atcagcgtct ggctgctggc ggtggcgatg aaatcgctgc ccctgggcac cgcctacgcg 180
gtgtgggtgg gcatcggcac gatcggcgcc ttcgtggccg gcatcgtgct gttcggtgaa 240
tcgaccagct ggatgcgcat cgccagcgtg gctctcatcg tgctcggcct ggtcggcctg 300
aagctgtcct cggcctga 318
Claims (9)
1. The resistance gene of chloramphenicol is characterized in that the gene sequence is shown in SEQ ID No.1 and is named as ykkC _ like gene.
Use of an inhibitor of ykkC _ like gene expression in the preparation of a product for reducing resistance of a subject to chloramphenicol.
3. Application of a reagent for detecting a SEQ ID No.1 sequence in preparing a kit for detecting chloramphenicol drug resistance.
4. The use of claim 4, wherein the reagent for detecting the sequence of SEQ ID No.1 is a reagent constructed by performing qualitative or quantitative detection on the sequence of SEQ ID No.1 by a PCR method.
Use of the ykkC _ like gene as a target gene for increasing the sensitivity of a subject to chloramphenicol.
6. The use according to any one of claims 2 to 5, wherein the chloramphenicol resistance is resistance to chloramphenicol in gram-positive and gram-negative bacteria.
7. A method for detecting chloramphenicol resistance is characterized in that the chloramphenicol resistance is detected based on SEQ ID No.1 sequence information.
8. The method of claim 7, wherein the method is not for diagnostic purposes.
9. The method of claim 7, wherein the chloramphenicol resistance is resistance to chloramphenicol in gram-positive and gram-negative bacteria.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111502A (en) * | 2020-09-25 | 2020-12-22 | 清华大学深圳国际研究生院 | Novel resistance gene of chloramphenicol and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112111502A (en) * | 2020-09-25 | 2020-12-22 | 清华大学深圳国际研究生院 | Novel resistance gene of chloramphenicol and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| ANDREIA CRUZ ET AL.: "sugE: A gene involved in tributyltin (TBT) resistance of Aeromonas molluscorum Av27", 《J. GEN. APPL. MICROBIOL.》 * |
| JACK, D L ET AL.: "A broad-specificity multidrug efflux pump requiring a pair of homologous SMR-type proteins", 《JOURNAL OF BACTERIOLOGY》 * |
| STRNAD,H.ET AL: "quaternary ammonium compound-resistance protein SugE 1 [Achromobacter xylosoxidans A8]", 《GENBANK，ACCESSION NO.ADP15474.1》 * |
| 张永，唐英春: "病原菌质子驱动型外排泵分子机制研究进展", 《国外医药抗生素分册》 * |
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