CN106167802A - A kind of novel integron In1290 - Google Patents

A kind of novel integron In1290 Download PDF

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CN106167802A
CN106167802A CN201610329148.9A CN201610329148A CN106167802A CN 106167802 A CN106167802 A CN 106167802A CN 201610329148 A CN201610329148 A CN 201610329148A CN 106167802 A CN106167802 A CN 106167802A
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integron
bacterial strain
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王冬国
周冬生
陈佳玉
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
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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
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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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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Abstract

The invention discloses a kind of novel integron In1290, its sequence is as shown in SEQ ID NO.1.This integron is to identify through enzyme action order-checking to obtain in the plasmid of a strain e coil k 1 pneumonia Resistant strain.Containing multiple drug resistant gene boxes in this integron, therefore the clinical application that is found to be of the above-mentioned integron of the present invention provides certain directive function, be conducive to reducing clinically the use of some antibacterials, reduce the selection pressure of such drug resistant gene box horizontal transfer, prevent the Outbreak of drug-resistant bacteria.

Description

A kind of novel integron In1290
Technical field
The invention belongs to Protocols in Molecular Biology and drug-resistant bacteria monitoring field, relate to a kind of newfound and kerekou pneumonia The primary closely-related integron of Salmonella drug resistance.
Background technology
The progress of medical level, miscellaneous antibiotic is day by day developed and puts on market.It is widely used in and faces Bed and veterinary's aspect, even in animal husbandry, the most often use it to promote growth, the just extensive application of these antibiotic, especially Being to use unreasonably, cause the drug resistance of antibacterial so that multidrug resistant problem is day by day serious, resistance mechanism is increasingly sophisticated.People are pre- Material is likely to be due to the appearance that gene mutation can cause the bacterial strain of the antibiotic of resistance to single, but occurs multiple in same bacterial strain simultaneously The drug resistance of antibiotic, very difficult gene mutation explains.Research proves, hereditary material water in strain and between strain Flat pass and broadcast, be the antibacterial important channel that obtains new drug resistance.Drug resistance is propagated the most widely and goes out between different strain Existing closely similar drug resistance mode, illustrates the importance of above-mentioned mechanism.Recent studies have shown that integron is at drug resistant gene The appearance of horizontal transmission and multiple antibiotic resistant strain plays an important role.
Integron (integron) is an energy by the intergrase identification of self coding, captures, integrate or shear cell Outer episome or genetic fragment, and it is allowed to be converted into the recombinant expression system of functioning gene.Become trapped and integrate thin The outer episome of born of the same parents or genetic fragment mostly are coding resistance protein such as beta-lactamase (β-lactamase), aminoglycoside Inactive enzyme, lincomycin class inactive enzyme, chloramphenicol acetyltransferase, Macrolide inactive enzyme, Aminoglycoside Acetylase (AAC), the drug resistant gene of Aminoglycoside phosphotransferase (ANT) nucleotidyltransferase (APH) etc..
Integron is in some cases by capturing favourable box gene, such as drug resistance gene box, and obtains survival advantage, But be also possible to capture some host is produced the box gene of toxicity and adversely affects, or excessively capture box gene and Breeding to Host Strains causes certain burden.Therefore, the integron of antibacterial is during capture alien gene box, thus it is speculated that one Surely there are complete set and fine regulatory mechanism, but this mechanism understands the most not yet.To the above-mentioned research about regulatory mechanism, It is beneficial to disclose the drug resistance of antibacterial, the especially multi-drug resistant of antibacterial.
Summary of the invention
The invention provides a kind of integron containing drug resistant gene box, by its named In1290, its sequence such as SEQ Shown in ID NO.1.This integron contain aadA16 box gene, dfrA27 box gene, arr-3 box gene, catB3 box gene and AacA4 box gene.Above-mentioned integron expresses multidrug resistant gene, thus causes the strain containing this integron to Multiple Classes of Antibiotics Medicine has resistance.These antibiotic include: aminoglycoside antibiotics, sulfa antibiotics, chloromycetin series antibiotics, grand sight Mycin class antibiotic and rifampicin class antibiotic.
Common aminoglycoside antibiotics includes: streptomycin, gentamycin, kanamycin, tobramycin, A meter Ka Star.
Common sulfa antibiotics includes: bacteresulf (SIZ), sulfadimidine (SM2), sulfadiazine (SD), sulfamethoxazole (SMZ), sulfamonomethoxine (SMM), sulfasalazine (SASP), mafenide (SML), sulfanilamide Pyrimidine silver, sulfacetamide (SA) and bactrim (trimethoprim+sulfamethoxazole).
Common chloromycetin series antibiotics includes: chloromycetin, CAPS.
Common spectinomycin class antibiotic includes: spectinomycin, miromycin, miramycin.
Common rifampicin class antibiotic includes: rifampicin, rifapentine.
The integron of the present invention is prepared by the following:
(1) in Taizhou municipal hospital infection section patient blood cultivates positive specimen, 1 strain is isolated to amikacin, celebrating The antibacterial of big mycin drug resistance；
(2) then the monoclonal bacterial strain separated (is mended by VITEK 2 and susceptibility flat board agar diffusion method in operation sequence Fill drug sensitive test paper) carry out Bacteria Identification and drug sensitive test, this bacterial strain identified is Klebsiella pneumonia, and it is named KP5325.Meanwhile, test confirms that this bacterial strain has notable resistance to Multiple Classes of Antibiotics；
(3) KP5325 strain culturing is expanded, extract the plasmid of described Resistant strain；
(4) plasmid after step (3) extraction purification is used BamHI enzyme action, agarose gel electrophoresis isolated one section The DNA fragmentation of a length of 5,238bp, is connected with pMD19-T carrier in conventional manner by this DNA fragmentation, the most heat-shock transformed method Proceed in competent cell E.coli TOP10, select positive transformant with blue white macula experiment, order-checking；
(5) utilize ContigExpress Program software to carry out sequence assembly, analyze its structure, identify sequence such as Sequence shown in SEQ ID NO.1.
The invention provides a kind of bacterial strain containing this integron.This Strain Designation is KP5325.The matter of KP5325 bacterial strain Containing integron In1290 in Li, its sequence is as shown in SEQ ID NO.1.
Present invention also offers a kind of recombiant plasmid containing integron In1290 sequence.Described plasmid comprises integron In1290 sequence.In the specific embodiment of the present invention, described recombiant plasmid is by integron In1290 sequence and pET32a Carrier restructuring forms, and integron insertion point is on pET32a carrier at BamHI restriction enzyme site.The sky that described recombiant plasmid uses Carrier can use any common plasmid vector to replace.The method of described recombiant plasmid is also well known to those skilled in the art Conventional method.
Present invention provides a kind of joint bacterial strain containing integron In1290.In order to study the drug resistance of integron, The present invention utilizes bacterial plasmid transduction experiment to carry out the function of Testing and appraisal integron In1290, by KP5325 bacterial strain and E.coli J53 AzR(to Hydrazoic acid,sodium salt drug resistance) bacterial strain co-cultures, by containing Hydrazoic acid,sodium salt (300mg/L) and amikacin (0.06mg/L) Plate screening conjugon, does drug sensitive test by conjugon, and recipient bacterium has the resistance of donor bacterium.Wherein, amikacin used can Replace with agents: tobramycin, kalamycin, gentamycin, bactrim, chloromycetin, rifampicin.
Present invention also offers a kind of recombinant bacterial strain containing integron In1290.For being further characterized by above-mentioned joint bacterial strain Resistance derive from the integron in KP5325 bacterial strain, this integron In1290 is cloned in pET32a carrier by the present invention, whole Zygote insertion point is on pET32a carrier at BamHI restriction enzyme site.And be transformed in wild type E.coli JM109, logical Cross the flat board containing amikacin and select positive colony (containing the antibacterial of pET32a-integron).Positive colony is done drug sensitive test, Result display KP5325 bacterial strain in integron In1290 restructuring E.coli JM109 bacterial strain can be made following Drug-resistant: Ah Meter Ka Xing, tobramycin, kalamycin, gentamycin, bactrim, chloromycetin, rifampicin, spectinomycin.
Advantages of the present invention and beneficial effect:
(1) present invention firstly discovers that the integron including multidrug resistant gene box that sequence is SEQ ID NO.1 In1290, the nucleotide sequence of this integron has no report in the prior art.
(2) discovery of integron sequence of the present invention, provides certain directive function for clinical application.Be conducive to clinically Reduce the use of some antibacterials, reduce the selection pressure of such drug resistant gene box horizontal transfer, prevent the quick-fried of drug-resistant bacteria The property sent out is popular.
Accompanying drawing explanation
Fig. 1 is the structural representation of the integron In1290 of the present invention.
Specific embodiment
Further illustrating the present invention below in conjunction with specific embodiment, embodiments of the invention are only used for explaining the present invention, It is not intended to limit protection scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition such as sambrook et al., Molecular cloning: retouched in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) The condition stated, or according to the condition proposed by manufacturer.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The qualification of embodiment 1 integron In1290
1, the separation of KP5325 bacterial strain and qualification
1.1 material
Bacterial susceptibility card: the AST-GN13 of France bio Merieux.AST-GN13 susceptibility kind has: amikacin, ammonia benzyl XiLin, ampicillin/sulbactam, aztreonam, cefazolin sodium, cefepime, cefotetan, ceftazidime, ceftriaxone, ring third Sha Xing, ertapenem, gentamycin, imipenum, levofloxacin, nitrofurantoin, piperacillin/Tazobactam Sodium, appropriate cloth are mould Element, SMZ.
Supplement drug sensitive test paper (experiment of susceptibility flat board agar diffusion): the scraps of paper derive from Oxoid company of Britain, have the new promise of compound recipe Bright (30 μ g), chloromycetin (30 μ g) and rifampicin (5 μ g).
1.2 method
Instrumental: municipal for Taizhou hospital infection section patient blood is cultivated positive bacterial strain transferred species and separates to blood plate Cultivate (at 35 DEG C containing 5%CO2Incubator is cultivated 16-18h), then by the pure antibacterial of separation in operation sequence at VITEK 2 Upper machine makees Bacteria Identification and drug sensitive test.
Supplement drug sensitive test: spread upon on MH flat board by the bacterium solution of 0.5 Maxwell unit, stick the new promise of compound recipe in operation sequence Bright, chloromycetin and the rifampicin scraps of paper, at 35 DEG C containing 5%CO2After 16-18h cultivated by incubator, identify by CLSI 2015 standard Drug sensitivity tests.
1.3 result
1.3.1 instrumental result
Medium sized gram negative bacilli, KIA produces acid/product acid, and glucose and lactose produce acid aerogenesis, oxidase the moon Property, VP is positive, and indole is negative, and citrate is positive, and power is negative, does not produce H2S, meets the characteristic of Klebsiella Pneumoniae, warp VITEK 2 microbiological analysis instrument is accredited as Klebsiella pneumonia, identifies that probability is 99%, by its named KP5325.
1.3.2 drug sensitive experiment result
Drug sensitive experiment result shows, KP5325 bacterial strain produces resistance for following medicine: amikacin, gentamycin, appropriate Obramycin, bactrim, SMZ, chloromycetin, rifampicin.
2, the separation of integron In1290 and qualification
2.1 method
2.1.1KP5325 the plasmid extraction of bacterial strain
The plasmid using TaKaRa extracts test kit on a small quantity, and operating procedure extracts bacteria plasmid DNA to specifications, as Pcr template ,-20 DEG C of Refrigerator stores are standby.
2.1.2 the acquisition of integron
(1) cleaning is dried and sterilized 0.5mL eppendorf pipe is numbered, be separately added into micropipette rifle KP5325 plasmid DNA 1 μ g and 10 × restriction enzyme reaction buffer 2 μ L (Fermentas), add redistilled water and make always Volume is 19 μ L, by adding 1 μ L BamHI enzyme liquid (Fermentas) after solution mixing in pipe, gets rid of with microcentrifuge, makes Solution concentrates at the bottom of pipe.
(2), after mixing reaction system, eppendorf pipe is placed on suitable holder and (is inserted on plastic foamboard), 37 DEG C of water bath heat preservation 2-3h, make endonuclease reaction complete.
(3) often pipe adds 2 μ L 0.1mol/L EDTA (pH8.0), and mixing, with stopped reaction.
(4) product after enzyme action is separated through agarose gel electrophoresis.
2.1.3 the qualification of integron
(1) DNA fragmentation of a length of 5,238bp through agarose gel electrophoresis isolated is reclaimed.
(2) use conventional method, the DNA fragmentation that step (1) reclaims is connected with pMD19-T carrier, the most heat-shock transformed Method proceeds in competent cell E.coli TOP10, selects positive transformant with blue white macula experiment, order-checking.
2.2 result
Sequencing result shows after being analyzed and splicing, and the DNA fragmentation of a length of 5,238bp belongs to I class integron, and this is whole Zygote includes the basic structure of I type integron and multiple box gene, sequence as shown in SEQ ID NO.1, structural representation such as Fig. 1 Shown in.
The function of embodiment 2 plasmid transduction experimentation integron In1290
1, method
(1) donor bacterium is KP5325 bacterial strain, and recipient bacterium is E.coli J53 AzR(to Hydrazoic acid,sodium salt drug resistance).By donor Bacterium, recipient bacterium are inoculated on LB flat board respectively, 35 DEG C of incubated overnight.The single bacterium colony of picking is inoculated in the LB meat soup of 4mL respectively, 37 DEG C of 220r/min shake bacterium and cultivate to exponential phase.Respectively take 0.5mL for, recipient bacterium in the LB meat soup of 4mL, 37 DEG C of static mistakes Night cultivates.Conjugon screens with pancreas soy agar (TSA) plate, containing Hydrazoic acid,sodium salt (300mg/L) and A meter Sha star in flat board (0.06mg/L).Put 35 DEG C and hatch 18-24h.Extract the plasmid (step is with embodiment 1) of Resistant strain, integrate with PCR method The detection of sub-In1290 sequence.
The primer of amplification integron In1290 sequence is as shown in table 1:
Table 1 primer sequence
The reaction system (50 μ L) of PCR: Mg2+2.5mmol/L, primer 30pmol/L, dNTP 0.2mmol/L, 2.5U Tap Enzyme and 2 μ L DNA profilings.
Amplification condition:
94 DEG C of denaturations 3min, then 94 DEG C of degeneration 1min, 52-58 DEG C of annealing 1min, 72 DEG C extend 1min, and 30 are followed Ring, last 72 DEG C extend to 10min.
By PCR primer through agarose gel electrophoresis analysis.DNA profiling place by a length of 5,238bp of amplified production Bacterial strain be referred to as integron In1290 engage shift successful positive strain.
(2) positive strain being carried out drug sensitive experiment, step is with embodiment 1.
1.2 result
Drug sensitive experiment result shows, integron In1290 sequence expresses the drug sensitive reaction of positive strain with KP5325 bacterial strain phase With, represent integron In1290 sequence joint and shift successfully, and this integron result in conjugative bacteria and creates agents Resistance: amikacin, gentamycin, tobramycin, SMZ, bactrim, chloromycetin, rifampicin.
The function of embodiment 3 gene recombinaton experimentation integron In1290
1, method
(1) PCR primer (nucleotides sequence is classified as SEQ ID NO.1) in embodiment 2 is connected with pMD19-T carrier, even Thing of practicing midwifery adds in the competence of 100 μ L E.coli JM109 and converts, and cultivates on the flat board containing IPTG, x-gal, Amp, IPTG, x-gal, Amp resistant strain extracts plasmid order-checking (step is with embodiment 1), and whether detection integron In1290 inserts In genome.Then integron In1290 sequence being cloned in pET32a carrier, the insertion point of integron carries at pET32a On body at BamHI restriction enzyme site, conventionally prepare, obtain the pET32a recombiant plasmid containing integron In1290 sequence. By in recombinant plasmid transformed to competence E.coli JM109 cell.
(2) the clone bacterium successfully constructed is carried out Resistance detection
Use France's VITEK 2 Bacteria Identification of bioMerieux and Analysis of Drug Susceptibility system and susceptibility flat board agar diffusion side Method (supplementing drug sensitive test paper) carries out Resistance detection to recombinant bacterium E.coli JM109 (pET32a-integron In1290).
2, result
Drug sensitive experiment result shows, the drug sensitive reaction of recombinant bacterium E.coli JM109 (pET32a-integron In1290) is same KP5325 bacterial strain is identical, represents integron In1290 sequence and is the most successfully incorporated in the genome of E.coli JM109, and should Integron result in recombinant bacterium E.coli JM109 and creates the resistance of agents: amikacin, gentamycin, appropriate cloth are mould Element, SMZ, bactrim, chloromycetin, rifampicin.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement And modification, these improve and modify also by the protection domain falling into the claims in the present invention.

Claims (9)

1. the integron containing drug resistant gene box, it is characterised in that described integron sequence is shown in SEQ ID NO.1.
2. a recombiant plasmid, it is characterised in that comprise the integron described in claim 1 in described recombiant plasmid.
Recombiant plasmid the most according to claim 2, it is characterised in that the load used in the preparation process of described recombiant plasmid Body constitution grain is pET32a carrier.
4. a conjugative bacteria, it is characterised in that containing the integron described in claim 1 in described conjugative bacteria；Described connect Closing antibacterial is by KP5325 bacterial strain and E.coli J53 AzRPrepared by bacterial strain.
5. a recombinant bacteria, it is characterised in that containing the integron described in claim 1 in described recombinant bacteria；Described heavy Group antibacterial is to proceed to the recombiant plasmid described in Claims 2 or 3 be prepared from E.coli JM109 bacterial strain.
6. the acquisition methods of the integron described in claim 1, it is characterised in that said method comprising the steps of:
(1) in Taizhou municipal hospital infection section patient blood cultivates positive specimen, 1 strain is isolated the most mould to amikacin, celebrating The antibacterial of element drug resistance；
(2) then the monoclonal bacterial strain separated (is supplemented medicine by VITEK 2 and susceptibility flat board agar diffusion method in operation sequence The quick scraps of paper) carry out Bacteria Identification and drug sensitive test, this bacterial strain identified is Klebsiella pneumonia, by its named KP5325. Meanwhile, test confirms that this bacterial strain has notable resistance to Multiple Classes of Antibiotics；
(3) KP5325 strain culturing is expanded, extract the plasmid of described Resistant strain；
(4) plasmid after step (3) extraction purification is used BamHI enzyme action, agarose gel electrophoresis isolated one segment length It is 5, the DNA fragmentation of 238bp, this DNA fragmentation is connected with pMD19-T carrier in conventional manner, the most heat-shock transformed method proceeds to In competent cell E.coli TOP10, select positive transformant with blue white macula experiment, order-checking；
(5) utilize ContigExpress Program software to carry out sequence assembly, analyze its structure, identify sequence such as SEQ Sequence shown in ID NO.1.
7. the preparation method of the conjugative bacteria described in a claim 4, it is characterised in that the concrete operations of described preparation method Step is as follows: by KP5325 bacterial strain and E.coli J53 AzRBacterial strain co-cultures, by containing Hydrazoic acid,sodium salt and KP5325 bacterial strain The plate screening conjugon of tolerance medicine.
Preparation method the most according to claim 7, it is characterised in that described KP5325 bacterial strain tolerance medicine is A meter Sha Star.
9. the preparation method of the recombinant bacteria described in a claim 5, it is characterised in that the concrete operations of described preparation method Step is as follows: be cloned in pET32a carrier by integron In1290, and then recombinant plasmid transformed enters wild type E.coli In JM109, select positive colony by the flat board containing amikacin.
CN201610329148.9A 2016-05-10 2016-05-10 A kind of novel integron In1290 Pending CN106167802A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913399A (en) * 2019-04-12 2019-06-21 贵州省水产研究所 A kind of Aeromonas media integron containing multiple drug resistant gene boxes and its acquisition methods and application

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450765A (en) * 2014-11-13 2015-03-25 王冬国 Integron In1069

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450765A (en) * 2014-11-13 2015-03-25 王冬国 Integron In1069

Non-Patent Citations (1)

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Title
GENBANK: "KM589496", 《GENBANK》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913399A (en) * 2019-04-12 2019-06-21 贵州省水产研究所 A kind of Aeromonas media integron containing multiple drug resistant gene boxes and its acquisition methods and application
CN109913399B (en) * 2019-04-12 2021-08-20 贵州省水产研究所 Aeromonas intermedia integron containing multiple drug-resistant gene cassettes and obtaining method and application thereof

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Application publication date: 20161130