CN112111502A - Novel resistance gene of chloramphenicol and application thereof - Google Patents
Novel resistance gene of chloramphenicol and application thereof Download PDFInfo
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Abstract
The invention provides a novel resistance gene of chloramphenicol and application thereof, wherein the resistance gene has a sequence shown in SEQ ID No.1 and is named as GMC gene, after third-generation sequencing of genome, no currently known drug-resistant gene is found on the genome, and then RNA sequencing is carried out to find that the oxidoreductase coded by GMC has high transcription expression amount under the action of antibiotics. The novel resistance gene discovers a potential drug resistance gene of the chloramphenicol, provides help for understanding a drug resistance mechanism of the chloramphenicol and supplementing a drug resistance database, and also provides a target for later drug resistance of the chloramphenicol.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a novel resistance gene of chloramphenicol and application thereof.
Background
The chloramphenicol antibiotic can act on 50S subunit of bacteria ribonucleoprotein body to inhibit protein synthesis, and belongs to antibacterial broad-spectrum antibiotic. The 70S ribosome of bacterial cells is the major cellular component for protein synthesis and comprises two subunits, 50S and 30S. Chloramphenicol reversibly binds to the 50S subunit, blocks the action of transpeptidylase, and interferes with the binding of the amino acid-bearing aminoacyl-tRNA terminal to the 50S subunit, thereby preventing the formation of new peptide chain and inhibiting protein synthesis. Since chloramphenicol can also bind to 70S of human mitochondria, it can also inhibit protein synthesis of human mitochondria, and is toxic to human body. Chloramphenicol is considered a bacteriostatic antibiotic because it binds reversibly to the 70S ribosome, but it can also be bactericidal against certain bacteria at high drug concentrations and bactericidal against influenza bacteria even at lower concentrations.
Chloramphenicol has inhibitory effect on gram-positive and gram-negative bacteria, and has strong effect on the gram-positive and gram-negative bacteria. Among them, it has stronger action on typhoid bacillus, influenza bacillus, parainfluenza bacillus and bordetella pertussis than other antibiotics, and is also effective on rickettsia infection such as typhus, but has no effect on gram-positive cocci as compared with penicillin and tetracycline. The antibacterial action mechanism is that the antibacterial peptide is combined with a 50S subunit of a ribosome to inhibit peptide acyltransferase, so that protein synthesis is inhibited.
Various bacteria can generate drug resistance to chloramphenicol, wherein escherichia coli, dysentery bacillus, proteus bacillus and the like are more common, and typhoid bacillus and staphylococcus are less common. Bacteria are relatively slow to develop resistance to chloramphenicol, probably through gradual mutation of the gene, but can automatically disappear. Bacteria can also acquire resistance by transfer of the R factor, which produces chloramphenicol acetyl transferase (acetyltransferase) to inactivate chloramphenicol.
Disclosure of Invention
Aiming at the background technology, the invention aims to provide a novel resistance gene of chloramphenicol, and the invention discovers a novel drug resistance gene GMC gene of chloramphenicol from sphingomonas, supplements a chloramphenicol resistance database and lays a foundation for resisting bacteria.
The first aspect of the invention provides a novel resistance gene, the sequence of which is shown as SEQ ID No.1 and is named as GMC gene.
In a second aspect, the present invention provides a recombinant vector comprising the novel resistance gene of the first aspect.
In a third aspect, the present invention provides a transformant containing the novel resistance gene of the first aspect, or the recombinant vector of the second aspect.
The third aspect of the invention provides an application of GMC gene of sphingomonas as drug resistance gene, wherein the GMC gene sequence is shown as SEQ ID No.1, and the drug resistance refers to resistance to chloramphenicol antibiotics.
Compared with the prior art, the invention has the beneficial effects that:
the novel resistance gene discovers a potential drug resistance gene of the chloramphenicol, supplements a chloramphenicol drug resistance database, provides help for understanding the drug resistance mechanism of the chloramphenicol, provides a target spot for scientific researchers to solve the chloramphenicol drug resistance, and lays a foundation for resisting bacteria drug resistance.
Drawings
FIG. 1 is a schematic diagram of the pBBR1MCS-2 plasmid;
FIG. 2 is a flow chart of the recombinant plasmid construction of pBBR1 MCS-2-GMC;
FIG. 3 is a diagram showing the alignment result of blast sequence on NCBI for successful construction of gene GMC.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments. The scope of the invention is not limited to the specific embodiments.
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The sphingomonas is found to have high resistance to chloramphenicol from the environment, no currently known resistance gene is found on the genome after the third-generation sequencing of the genome, and then the sequencing of RNA shows that the oxidoreductase coded by GMC has high transcription expression level under the action of antibiotics.
In order to study the function of the novel resistance gene GMC gene of chloramphenicol in detail, the present invention includes the following examples:
example 1
A method of material
1. Selection of appropriate vectors
The pBBR1MCS-2 plasmid was selected and shown in FIG. 1 as a schematic diagram of the pBBR1MCS-2 plasmid, which is a broad host protein expression and cloning plasmid and has the characteristics of high copy, multiple cloning sites, stable expression, constitutive plasmid and the like.
2. Design of enzyme digestion primer
Upstream primer
Downstream primer
TCCCCCGGGCTGCAGGAATTCTCAGTGGCTTCTTCGGATCA
GAATTCRepresents an EcoRI cleavage site,
shows an SD sequence having a function of enhancing gene expression,
indicating an additional start codon sequence to prevent the inserted foreign sequence gene from replicating.
The promoter sequence is lac promoter carried by plasmid pBBR1-MCS2, tttacactttatgcttccggctcgtatgttg
3. Construction of recombinant vectors
3.1 acquisition of the Gene of interest GMC
Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying a specific DNA fragment, and can greatly increase a trace amount of DNA. The test adopts a 20 mu L reaction system, 20ul genome DNA of the PCR reaction system is a gold and silver group DNA extracted from sphingomonas by using a small radix-rooted genome kit, and is a 20 mu LPCR reaction system as shown in Table 1, and the reaction program is as follows: the reaction procedure is as follows:
and after the reaction is finished, storing the product in a refrigerator at 4 ℃.
TABLE 120 μ L PCR reaction System
3.2pBBR1MCS-2 plasmid extraction
Bacterial escherichia coli DN5 alpha of pBBR1MCS-2 plasmid is adopted, the strain is stored in a laboratory, the strain is inoculated into 5mL of LB culture solution, after shaking culture is carried out at 37 ℃ and 180rpm overnight for activation, 100L of bacterial solution is sucked in a super clean bench and inoculated into a new LB culture solution test tube, shaking culture is carried out at 37 ℃ and 180rpm, and subsequent operation can be carried out after the bacterial solution is not clear any more after 3 hours.
The plasmid pBBR1MCS-2 was extracted using a plasmid miniprep kit manufactured by Tiangen Biochemical technology Ltd as follows.
3.3 Single cleavage of vector and target Gene
The pBBR1MCS-2 plasmid has a series of restriction enzyme cutting sites of different restriction enzymes, including EcoRI restriction enzyme cutting site, and the self-replication of the plasmid is not influenced after the cutting. The target gene GMC gene has introduced specific restriction site of restriction endonuclease EcoRI into the primer designed during PCR amplification. Therefore, the two ends of the target gene can be specifically matched with the gaps of the plasmids after single enzyme digestion, which lays a foundation for the next connection.
A50-mu-L reaction system is adopted for single enzyme digestion reaction of the target gene GMC gene and the vector, as shown in Table 2, after sample addition, a pipette is used for gently blowing and beating the mixture evenly, the mixture is placed in a water bath kettle at 37 ℃ for enzyme digestion reaction, and the mixture is taken out after 4 hours.
TABLE 250 μ L Single enzyme digestion reaction System
3.4 recovery
Adding a LoadingBuffer with the final concentration of 1X into the enzyme digestion product, gently blowing and uniformly mixing by using a pipette gun, carrying out agarose gel electrophoresis on all the enzyme digestion products, and respectively recovering highlight bands cut off on an electrophoresis gel block under an ultraviolet lamp by using a DNA purification and recovery kit produced by Tiangen (Tiangen) Biotechnology limited company.
3.5 connection
The construction of recombinant vectors by linking the vectors with the target gene GMC gene is the basis for the large-scale expression of the target gene in the expression strains. The specific operation of the connection is as follows: after the concentration of the recovered product is measured, the addition amount of each component in the system is calculated according to a molar ratio formula of the connected product. The test shows that the concentration of GMC after double digestion is 84.3 ng/. mu.L, and the concentration of pBBR1MCS-2 after single digestion is 46.1 ng/. mu.L.
Four groups of parallel tests were performed using a 10. mu.L system, the ligation system is shown in Table 3, the product was sequentially added to a 250. mu.L vial, gently blown and mixed with a pipette, and then placed in a refrigerator at 4 ℃ overnight to complete the ligation reaction.
TABLE 3 ligation product 10. mu.L
3.6 transformation
3.6.1 preparation of DH5 alpha competent cells
The recombinant vector is transformed into an active strain to enable self-replication, and therefore the bacteria are first rendered competent to receive the exogenous plasmid. Coli DH 5. alpha. was used as a site for replication of the recombinant vector in this experiment, and DH 5. alpha. competent cells were prepared by the TSS method. The specific operation steps are as follows:
(a) preserving glycerol strain at-50 deg.C according to the ratio of 1: 100 proportion the overnight cultured broth was added to two triangular flasks containing 50mL of fresh LB medium with a volume of 250mL, and incubated at 37 ℃ with shaking (225rpm) until the pre-exponential phase (OD600 of about 0.3-0.4, incubation time 2h-2.5h) in LB medium as OD measurement blank.
(b) Centrifuging and precipitating, resuspending the cells in 1 XTSS solution, subpackaging and storing until each cell is transferred to 1 50mL clean centrifuge tube, cooling the culture to 0 ℃ in an ice bath for 15-30 minutes, centrifuging for 10min at 1000g at 4 ℃, discarding the supernatant, and harvesting the bacteria.
(c) 5mL of 1 XTSS solution (ice pre-cooled for 30min) is added to suspend the cells, and the cells are kept on ice for 5-15min to prepare competent cells. Then subpackaging 100 μ L/portion into pre-cooled 1.5mL centrifuge tubes for transformation, and freezing in a refrigerator at-50 deg.C.
3.6.2 transformation of DH 5. alpha. competent cells
Transformation of a transformant strain which can be introduced with the recombinant vector into a recipient bacterium and then contains the desired DNA insert can be carried out by various methods, and this experiment uses the KCM method for transformation. The method comprises the following specific steps:
(a) placing the EP tube containing the competent cells on ice to melt the competent cells;
(b) the centrifuge tube of 1.5mL is removed again, 20. mu.L of 5 XKCM, 5. mu.L of recombinant plasmid and ddH are added2O to 100 mu L, mixing evenly and placing in ice;
(c) slowly and uniformly adding 100 mu L of competent fine powder into the EP tube, gently blowing and beating the mixture uniformly, and continuously standing the mixture on ice for 30 min;
(d) heat shocking at 42 deg.C for 90s, and inserting into ice for 30min
(e) Adding 1mL LB culture solution preheated at 37 deg.C, placing on a shaker at 37 deg.C for culturing at 90rpm for 20min, and 200rpm for 40 min;
(f) 100 μ L of the bacterial suspension was inoculated into a kan-containing LB plate or centrifuged at 8000rpm for 1min, and the bacteria were resuspended in preheated LB plate, and then inoculated into a kan-containing LB plate and cultured in an inverted state in a 37 ℃ incubator overnight.
In addition, CK2 was coated with untransformed DH 5. alpha. competent cells and CK3 was transformed E.coli DH 5. alpha. with the kan-resistant LB vector as CK1 group (i.e., blank control group), and pBBR1MCS-2 vector as empty vector.
The recombinant vector constructed, i.e., the recombinant plasmid of pBBR1MCS-2-GMC, is shown in FIG. 2.
3.6.4 authentication
The transformation was carried out to construct E.coli DH 5. alpha. strain containing the gene of interest GMC, and thus the results of the transformation were examined in various ways to determine whether the E.coli DH 5. alpha. strain containing the gene of interest GMC was successfully constructed. Among them, it is most convincing that the constructed strain contains the sequence of the target gene GMC. Table 4 shows the blast alignment result on NCBI, and it can be seen from table 4 that the two sequences are identical, the gene GMC is successfully constructed, the blast sequence alignment result on NCBI that the gene GMC is successfully constructed is shown in fig. 3, and the aligned sequence information can be seen in fig. 3.
TABLE 4 blast sequence alignment results
| Max Score | Total Cover | Query Cover | E value | | Accession | |
| 2964 | 2964 | 100.00％ | 0.0 | 100.00％ | Query_57447 |
MIC verification of Gene function
After the recombinant vector was electrically transformed into Pseudomonas putida KT2 and the MIC value of the Minimal Inhibitory Concentration (MIC) value to chloramphenicol was determined, it was found that the MIC value to chloramphenicol of KT2 strain containing pBBR1MCS-2-GMC + plasmid was 240ug/ml, whereas that to chloramphenicol of KT2 strain containing no pBBR1MCS-2-GMC + plasmid was 2 ug/ml. Indicating that the redox enzyme gene encoded by GMC makes the strain resistant to chloramphenicol, and GMC is also a potential drug resistance gene of chloramphenicol.
Although only the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art, and all changes are encompassed in the scope of the present invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
SEQUENCE LISTING
<110> Shenzhen International institute for graduate of Qinghua university
<120> novel resistance gene of chloramphenicol and use thereof
<130> 2020
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1605
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<213> genus Sphingomonas
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aagcagctca tcgaaggatg ccggatcgtg cgcaagattt tccgttccaa gccattcagt 1320
gaatatgaca aaggtgaacg cttacccgga aagcaggtcg aaaccgacgc tgattggatc 1380
gagtatatcc gtcagagcgc cttcctgatg taccacccga ctggcacttg cgcgatggga 1440
attgggccga cagcggttct cgatccggag ttgcgcgtca agggcgtcac cggtcttcgc 1500
gttgcggatg cctcgatcat gccgacgctg gttagcgcga atacaaatgc accgtgcatc 1560
atgattggcg aacgggcggc cgatctgatc cgaagaagcc actga 1605
Claims (5)
1. The novel resistance gene is characterized in that the sequence of the novel resistance gene is shown as SEQ ID No.1 and named as GMC gene.
2. A recombinant vector comprising the novel resistance gene of claim 1.
3. A transformant containing the novel resistance gene according to claim 1 or the recombinant vector according to claim 2.
4. A method for producing a transformant, characterized by comprising the steps of: introducing the novel resistance gene according to claim 1 or the recombinant vector according to claim 2 into a host.
5. The GMC gene is shown in SEQ ID No.1, and the drug resistance refers to resistance to chloramphenicol antibiotics.
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| CN113151526A (en) * | 2021-06-07 | 2021-07-23 | 清华大学深圳国际研究生院 | PCR detection method of novel chloramphenicol resistance gene from GMC oxidase family |
| CN113293201A (en) * | 2021-06-23 | 2021-08-24 | 清华大学深圳国际研究生院 | Rapid quantitative detection method of novel chloramphenicol resistance gene and application thereof |
| CN113337591A (en) * | 2021-06-30 | 2021-09-03 | 清华大学深圳国际研究生院 | Method for quantifying activity of antibiotic resistance gene in environment based on macrotranscriptomics and macrogenomics and identifying host |
| CN113403328A (en) * | 2021-07-13 | 2021-09-17 | 清华大学深圳国际研究生院 | Novel ykkkkKD resistance gene of chloramphenicol and application thereof |
| CN113416740A (en) * | 2021-07-13 | 2021-09-21 | 清华大学深圳国际研究生院 | Novel ykkC resistance gene of chloramphenicol and use thereof |
| CN113462698A (en) * | 2021-06-30 | 2021-10-01 | 清华大学深圳国际研究生院 | Novel resistance gene of lincomycin and application thereof |
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| CN113151526A (en) * | 2021-06-07 | 2021-07-23 | 清华大学深圳国际研究生院 | PCR detection method of novel chloramphenicol resistance gene from GMC oxidase family |
| CN113293201A (en) * | 2021-06-23 | 2021-08-24 | 清华大学深圳国际研究生院 | Rapid quantitative detection method of novel chloramphenicol resistance gene and application thereof |
| CN113337591A (en) * | 2021-06-30 | 2021-09-03 | 清华大学深圳国际研究生院 | Method for quantifying activity of antibiotic resistance gene in environment based on macrotranscriptomics and macrogenomics and identifying host |
| CN113462698A (en) * | 2021-06-30 | 2021-10-01 | 清华大学深圳国际研究生院 | Novel resistance gene of lincomycin and application thereof |
| CN113481309A (en) * | 2021-06-30 | 2021-10-08 | 清华大学深圳国际研究生院 | Method for high-sensitivity rapid quantitative detection of novel chloramphenicol resistance gene in environmental sample and application thereof |
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| CN113403328A (en) * | 2021-07-13 | 2021-09-17 | 清华大学深圳国际研究生院 | Novel ykkkkKD resistance gene of chloramphenicol and application thereof |
| CN113416740A (en) * | 2021-07-13 | 2021-09-21 | 清华大学深圳国际研究生院 | Novel ykkC resistance gene of chloramphenicol and use thereof |
| CN113512603A (en) * | 2021-07-13 | 2021-10-19 | 清华大学深圳国际研究生院 | Method for detecting new resistance genes in environmental sample based on Nanopore metagenome sequencing |
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