CN106566830A - SiRNA for regulating expression of integrase and drug-resistant gene cassette and application thereof - Google Patents
SiRNA for regulating expression of integrase and drug-resistant gene cassette and application thereof Download PDFInfo
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- CN106566830A CN106566830A CN201610961933.6A CN201610961933A CN106566830A CN 106566830 A CN106566830 A CN 106566830A CN 201610961933 A CN201610961933 A CN 201610961933A CN 106566830 A CN106566830 A CN 106566830A
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C12N2310/00—Structure or type of the nucleic acid
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Abstract
The invention discloses an siRNA for regulating expression of integrase and a drug-resistant gene cassette and an application thereof. A drug-resistant bacteria strain is introduced into the designed siRNA, and capture of an integrase gene on the drug-resistant gene cassette and expression of a drug-resistant gene in an integrated subsystem are silenced and interfered. Through RT-PCR quantitative detection of changes of the integrase gene mRNA expression level, microbial drug-resistant detection, integrase integration efficiency detection and other methods, the interference efficiency on the siRNA is evaluated, and the pathogenic microorganism drug-resistant problem caused by integron is controlled and eliminated by the siRNA ultimately. The expression of the integrase and the drug-resistant gene can be effectively and specifically interfered to inhibit bacterial drug resistance, and a new idea is widened for development of new drugs.
Description
Technical field
The present invention relates to a kind of for expression regulation factor siRNA of intergrase and drug resistant gene box and its application.
Background technology
Foodsafety and food source microbiological pollution, remain ancient in Food Science and challenging class
Topic.So-called microorganism drug resistance refers to that the antibacterials of certain concentration to generally suppressing its growth and breeding generate tolerance
Property.This concept is suggested in the 1950's, away from first antibiotic --- penicillin clinical practice only 4 year.
Carrying the antibacterial of drug-resistance factor can be propagated by fertilizer, feedstuff and food etc. between animals and plants, microorganism and the mankind, but drug resistance
It is considered as the main cause that causes human disease's bacterium drug resistance that property is broadcast to the mankind by food chain.The mankind are by constantly developing
New antimicrobial agent is accompanied by the clinical practice of new antimicrobial agent suppressing and eliminate the growing complicated drug resistance of antibacterial
New Resistant strain also begins to be occurred.The continuous propagation of bacterial resistance and can not the property eliminated so that it is to human survival health and production
Life causes high risks.The main biochemical mechanism of bacterial resistance includes：(1) microorganism is by the way that producing enzyme is to antibiotics modification or breaks
It is bad；(2) antibiotic is hindered to permeate into antibacterial；(3) antibacterial active efflux mechanism；(4) Target alterations or new target position are produced.Antibacterial
The biochemical mechanism of drug resistance is carried out under the guidance of bacterial resistance gene mechanism[2].In the past with regard to bacterial drug resistance genes mechanism machine
The study limitation of reason in gene mutation and by plasmid-mediated two aspects of drug resistance, but with extremely low bacterial gene mutation rate
The fast development and universality that drug resistance is explained with plasmid-mediated unstability is not definite enough.Recently, with capture
And the integration subsystem of expression alien gene ability is just increasingly causing the concern of researchers as bacterial drug resistance mechanism.
Integron is by the int I genes of an encoding integrase (integrase), two gene recombinaton site att I
Constitute with attC and promoter.Intergrase plays an important role in integron, and intergrase can be catalyzed drug resistant gene box
Integrate and reject so that drug resistant gene box is recombinated in different integrons.It is present in various bacteria due to integrating minor structure
In, particularly pathogenic bacterium and clinical strains, intergrase different drug resistant genes is propagated in antibacterial of the same race and different bacterium and by
The drug resistance of antibacterial is gradually improve, causes antibacterial to be evolved to drug resistance direction, very big potential hazard is caused to human health, therefore,
The expression regulation factor of intergrase and drug resistant gene box is studied, the method for finding silence its gene expression is conciliate to control
The resistance problems that certainly integron causes have great theoretical and using value.
Gene silencing mainly has two kinds of situations：A kind of is the gene silencing (transcriptional on transcriptional level
Genesilencing, TGS)；Another kind is PTGS (post-transcriptional gene
Silencing, PTGS), RNA interference (RNA interference, RNAi) is posttranscriptional gene resistance developed in recent years
Disconnected technology[7].RNA interference refers to that double-stranded RNA (double-strand RNA, dsRNA) is specifically induced in the cell therewith
The degraded of the mRNA of homologous complementary, closes the expression of corresponding gene, so as to initiating radical is because of the phenomenon of PTGS[8].Though
So in the organic difference for having detail in vivo of difference, but the mechanism of action of RNAi is highly conserved, generally comprises two ranks
Section：(1) initial period：Endogenouss or exogenous dsRNA recognize and are cut into small molecule of the segment size in 21-23nt by enzyme
RNA interfering (small interfering RNA, siRNA)；(2) effective stage：SiRNA is combined with nuclease complex, is formed
RNA inductions silencing complex (RNA-induced silencing complex, RISC), the latter has endonuclease activity,
The degraded siRNA of specificity recognizes homologous said target mrna by base complementrity principle.RNAi is a kind of very effective reduction gene
Expression, but in early stage, RNAi applications are limited in plant and animal C. Elegans Automatic Screening and fruit bat, and this is due in these biologies
Long-chain dsRNA can effective induced gene specificity silence.Being longer than the dsRNA of 30nt in mammal can suppress undifferentiated cell
The expression of middle specific gene, and PKR, IFNs can be produced by the mechanism induction of activation antiviral defense in noble cellss
Deng, cause rna transcription product nonspecific degradation and albumen synthesis stop completely.Subsequently this problem is by directly external synthesis
21nt dsRNA effect intermediate --- siRNA and broken through.Short chain siRNA can suppress to react with induced gene specificity,
Host's ifn response is avoided that again.This discovery causes siRNA technologies extensively to be applied in mammal.RNAi has
The advantages of high specific, high speed, high stability, high efficiency.Early stage researcher thinks that always RNAi phenomenons exist only in eucaryon
Biology, Japanese scholars Katsunori et al. suppresses staphylococcus aureuses blood coagulation with the siRNA of chemosynthesis at the beginning of 2006
After expression of enzymes, people just recognize that RNAi mechanism is existed in prokaryote.China is antibiotic usage big country, control and
Bacterial resistance issue concerns are solved to human survival health and economic development.By taking the change of Drug Resistance of E. coli as an example, to mistake
Remove in decades colibacillary drug in food, data display, Drug Resistance of E. coli is in gradually increasing in food
Trend.
The content of the invention
In order to solve the above-mentioned amount of asking, the invention provides the sequence of small molecules interference RNA (siRNA), can be by this
A little siRNA import integrase gene in Resistant strain silences and interference integron to the capture of drug resistant gene box and drug resistant gene
Expression.
It is an object of the invention to provide the siRNA of the expression for regulating and controlling intergrase and drug resistant gene box and its application.
The technical solution used in the present invention is：
For regulating and controlling the siRNA of intergrase and the expression of drug resistant gene box, the siRNA is siRNA-I and siRNA-II；Institute
The sequence for stating siRNA is respectively：
siRNA-I：
SiRNA-I positive-sense strands：5’-GCUGAAAGGUCUGGUCAUATT-3’(SEQ ID NO：1),
SiRNA-I antisense strands：5’-UAUGACCAGACCUUUCAGCTT-3’(SEQ ID NO：2),
siRNA-II：
SiRNA-II positive-sense strands：5’-CCCGUUCCAUACAGAAGCUTT-3’(SEQ ID NO：3),
SiRNA-II antisense strands：5’-AGCUUCUGUAUGGAACGGGTT-3’(SEQ ID NO：4).
Applications of the siRNA described above in drug-resistant microorganism drug resistance is reduced.
Further, drug-resistant microorganism described above is the drug-resistant microorganism containing integrase gene and drug resistant gene box.
Further, drug-resistant microorganism described above is the drug resistance large intestine bar containing integrase gene and drug resistant gene box
Bacterium.
Further, integrase gene described above is selected from intI1, intI2, intI3.
Further, drug resistant gene box described above selected from aad, aac, dfr, bla, oxa, cat, ere, sat, grr,
str。
Applications of the siRNA described above in overriding resistance microbial reagent is prepared.
The invention has the beneficial effects as follows：
1) present invention takes the lead in solving the basic (table of intergrase and drug resistant gene box of bacterial drug resistance using RNAi technology
Up to), by siRNA of the invention specific, efficiently can specifically disturb the expression of intergrase and drug resistant gene to reach suppression
Bacterial drug resistance processed, is the open new thinking of new drug development.
2) present invention solves difficulty of the RNA perturbation techniques in prokaryotic micro-organisms practical operation, is the extensive of the technology
Using blaze the trail.
3) based on the drug resistance pathogenic microorganism of the mediated by integron that the present invention is had been detected by by this laboratory, by setting
Count specific small molecules interference RNA (siRNA) interference integrase gene expression and the expression of drug resistant gene box.By present invention design
SiRNA imports drug resistance bacteria strain, and capture and drug resistance of the integrase gene to drug resistant gene box in subsystem is integrated in silence and interference
The expression of gene.By the change of RT-PCR detection by quantitative integrase gene mRNA expressions, microorganism Resistance detection and
The methods such as intergrase integration efficiency detection are estimated to the jamming effectiveness of siRNA.It is finally reached and is controlled by siRNA of the present invention
The pathogenic microorganism resistance problems caused with elimination integron.The present invention inquires into the regulation and control of pathogenic microorganism drug resistance from gene level
Mechanism, its result of study is not only facilitated and understood by the pathogenic microorganism drug resistance genetic background of mediated by integron, and is alternatively
RNA perturbation techniques are applied to prokaryote and exploitation novel antibacterial medicine provides scientific basis.
Description of the drawings
Fig. 1 is the collection of illustrative plates of pGPU6siRNA expression vectors.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1, siRNA
The present invention finally found that 2 kinds of siRNA are whole to silence and interference pathogenic bacteria by the substantial amounts of experimentation of early stage and screening
Integrase gene has preferable effect (efficiently special to the capture of drug resistant gene box and the expression of drug resistant gene in zygote system
Property), the particular sequence of this 2 kinds of siRNA is as described below.
siRNA-I：
SiRNA-I positive-sense strands：5’-GCUGAAAGGUCUGGUCAUATT-3’(SEQ ID NO：1),
SiRNA-I antisense strands：5’-UAUGACCAGACCUUUCAGCTT-3’(SEQ ID NO：2),
siRNA-II：
SiRNA-II positive-sense strands：5’-CCCGUUCCAUACAGAAGCUTT-3’(SEQ ID NO：3),
SiRNA-II antisense strands：5’-AGCUUCUGUAUGGAACGGGTT-3’(SEQ ID NO：4).
The synthesis of above-mentioned siRNA can be directly synthesized (can be synthesized by corresponding biotech firm) by chemical method.To studying
The problem being likely to occur in journey takes following method to solve.First, transfecting siRNA can only obtain of short duration inhibition, may
It is degraded due to RNA and the dilution of siRNA concentration.To avoid the Degradation of RNase, the nucleotide using modification is favourable
Stablizing so as to improving transformation efficiency and extending the depression effect time in siRNA.Next to that transfection problems, external acquisition
SiRNA can pass through the methods such as classical liposome, electroporation, microinjection and import cell, and this research plan takes liposome bag
The method of conjunction.
The structure of embodiment 2siRNA interference carrier
1) siRNA annealing
Synthetic embodiment 1siRNA positive-sense strand and antisense strand TE are dissolved into into the solution that concentration is 100 μM, according to
Following table prepares annealing reaction system.
Above-mentioned annealing reaction system is placed in PCR instrument and is made annealing treatment：95 DEG C, 5min；85 DEG C, 5min；75 DEG C,
5min；70 DEG C, 5min；4 DEG C are stored in, shRNA fragments are obtained.
2) structure of pGPU6/GFP/Neo-siRNA expression vectors
PGPU6/GFP/Neo (being purchased from Shanghai JiMa pharmacy Technology Co., Ltd) Vector maps are as shown in figure 1, by pGPU6/
GFP/Neo carriers obtain purpose linear fragment Jing after Bbs I and the enzyme action of BamH I, then by the shRNA fragments and enzyme action of above-mentioned gained
PGPU6/GFP/Neo afterwards is attached, and linked system is as follows：
22 DEG C of reactions convert the competent cells of escherichia coli JM 109 after 1 hour；By the recombinant bacterial strain of acquisition with containing
The LB culture medium of Kanamycin is screened, picking positive colony bacterium colony, and extracting plasmid carries out enzyme action and sequence verification, verifies
It is correct to be required siRNA interference carriers.The interference that siRNA-I and siRNA-II is obtained respectively according to the method described above is carried
Body.
The detection of embodiment 3siRNA jamming effectiveness
Experimental technique：
In order to detect the jamming effectiveness of siRNA of the present invention, respectively by the siRNA-I prepared in embodiment 2 and siRNA-II
Interference carrier transfects cause fastbacteria E. coli BL21 (DE3), and the intergrase that the antibiotic-resistance E. coli contains is
IntI1, the drug resistant gene box for containing is aadA1.Transfection method is：SiRNA is added in three times, is added once every 2h, altogether training
The siRNA of final concentration of 0.2 μM of foster system, the co-cultivation time is 8h.The fastbacteria after RT-PCR detection by quantitative siRNA before processings
The mRNA expressions of E. coli BL21 (DE3) target gene (intergrase IntI1, drug resistant gene box aadA1)；
Carry out similar process as a control group with nonsense sequence simultaneously.
Above-mentioned RT-PCR systems and program are：
Real-time fluorescence quantitative PCR reaction is carried out to target gene using Bio-Bad CFX 96Real-time PCR instruments, often
3, individual sample is parallel.The μ L of reaction system 25, wherein each reagent volume is：Premix Ex Taq II 1X 12.5μ
L；The each 1 μ L of upper primer, lower primer；The μ L of DNA profiling to be measured 2；ddH2O 8.5μL.Each sample carries out 3 technologies to be repeated to test, and 3
Secondary repetition amplifies then to be considered positive.
Course of reaction is：95 DEG C of denaturations 1min；95 DEG C of degeneration 15s, 60 DEG C of annealing 1min, 72 DEG C of extension 30s, 40 are followed
Ring；Automatic Program heats up and carries out melting curve analysis.
Experimental result：
Standard curve is done with RT-PCR, the mRNA expressions of target gene IntI1 and aadA1 before and after quantitative siRNA suppression,
As a result show and compare matched group, the colibacillary IntI1 of fastbacteria Jing after siRNA-I of the present invention and siRNA-II are processed with
AadA1 expressions are substantially reduced, and illustrate that expression of the siRNA of the present invention to integrase gene and drug resistant gene box has dry well
Disturb efficiency.
The detection of bacterial drug resistance after the interference of embodiment 4siRNA
Method：SiRNA-I the and siRNA-II interference carriers prepared in embodiment 2 are transfected into respectively cause fastbacteria large intestine bar
Bacterium E.coli BL21 (DE3), contains intergrase IntI1, containing drug resistant gene box aadA1, to amino in the antibiotic-resistance E. coli
Sugared tobramycin antibiotic spectinomycin, streptomycin and sulfa drugss trimethoprim have drug resistance.Will the transfection present invention
Antibiotic-resistance E. coli E.coli BL21 (DE3) after siRNA is cultivated in the culture medium containing spectinomycin, and detection is dry
Disturb the drug resistance of rear E. coli BL21 (DE3).Carry out similar process as a control group with nonsense sequence simultaneously.
As a result：Experimental result shows, transfects the antibiotic-resistance E. coli E.coli after siRNA-I and siRNA-II of the present invention
BL21 (DE3) can hardly grow in the culture medium containing spectinomycin, and in the matched group that nonsense sequence is processed, drug resistance is big
Enterobacteria E.coli BL21 (DE3) can in the culture medium containing spectinomycin normal growth.
Embodiment 5RT-PCR absolute quantitation detects siRNA integration rates
The present embodiment is with the drug resistant gene level effect of the mediations of Int I 1 before and after the interference of real-time PCR quick detections siRNA
Rate changes, and using different quantitative manners, many-side evaluates impacts of the siRNA to drug resistant gene horizontal transmission.Detection siRNA suppressions
System integrates the impact of expression of enzymes and siRNA to integration efficiency.
Method is as follows：Respectively by plasmid containing p184-2addA, the E.coli incubated overnight of the plasmids of pR 388,100 μ l are respectively taken
After mixed liquor, it is put in 1ml LB and co-cultures, and starts to add siRNA-I the and siRNA-II interference carriers that embodiment 2 builds,
Each hour adds a siRNA；Co-culture 8 hours；Then blank group, negative control group are determined with RT-PCR relative quantifications method
(nonsense sequence), the difference of middle intergrase mrna expression amount in siRNA groups of the present invention, RT-PCR absolute quantitation method determines blank group
With cointegrate in siRNA and the copy number and integration rate of original plasmid R388.
Quantitative fluorescent PCR absolute quantitation testing result shows：The resistance to of sample is processed through negative control group (nonsense sequence)
Medicine box gene integration efficiency is 6.9 × 105, and the drug resistant gene of the sample processed with siRNA-I of the present invention and siRNA-II
Box integration efficiency is respectively 3.9 × 106、3.1×106, illustrate that siRNA of the present invention can be played and suppress what drug resistant gene box was integrated
Effect.Illustrate that siRNA of the present invention can control and eliminate the pathogenic microorganism resistance problems that integron causes.
Intergrase integration efficiency detection in the drug-resistant bacteria of embodiment 6
Method：With integration efficiency as Testing index.Integration rate is that the drug resistant gene box after integrated sub- intergrase excision is whole
Close the efficiency on integron site.Its measure is related to supply, (is contained the E.coli of p184-2addA plasmids and containing pR by thalline
The E.coli of 388 plasmids) joint efficiency, the excision efficiency and the integration efficiency of integron of integron.The calculating of integration efficiency
Formula is as follows：
The computing formula of integration efficiency：Integration rate=(I1/N) × (M/D) × (V1/V2)
I1/N --- PCR positive rates；
M/D --- joint efficiency；
N --- picking single bacterium colony enters the number of performing PCR checking；
M --- TMP (trimethoprim), SUL (sulfonamides)+corresponding gene box resistant panel bacterium number；
D --- TMP, SUL resistant panel bacterium number；
V1 --- TMP, SUL resistant panel applies bacterium amount；
V2 --- TMP, SUL+ corresponding gene box resistant panel applies bacterium amount.
As a result：With int I 1 as target gene, by the special siRNA of present invention design, after co-culturing with donor bacterium and recipient bacterium,
The reduction of detection integration efficiency finds that the integration efficiency of siRNA-I, siRNA-II, feminine gender siRNA and water is respectively 3.2 × 10-6、3.7×10-4、6.5×10-4With 7.1 × 10-4Cfu/ (donor bacterium), siRNA-I suppression ratio corresponding with siRNA-II is respectively
54.1%th, 50.2%.
The above results illustrate that siRNA of the present invention can well suppress the integration efficiency of intergrase in drug-resistant bacteria, can be with
The pathogenic microorganism resistance problems that control and elimination integron cause.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Guangdong University of Petrochemical Technology
<120>For regulating and controlling siRNA and its application of intergrase and the expression of drug resistant gene box
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
gcugaaaggu cuggucauat t 21
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
uaugaccaga ccuuucagct t 21
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
cccguuccau acagaagcut t 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
agcuucugua uggaacgggt t 21
Claims (7)
1. the siRNA for regulating and controlling intergrase and the expression of drug resistant gene box is used for, and the siRNA is siRNA-And siRNA-；It is described
The sequence of siRNA is respectively：
siRNA-：
siRNA-Positive-sense strand：5’-GCUGAAAGGUCUGGUCAUATT-3’（SEQ ID NO：1）,
siRNA-Antisense strand：5’-UAUGACCAGACCUUUCAGCTT-3’（SEQ ID NO：2）,
siRNA-：
siRNA-Positive-sense strand：5’-CCCGUUCCAUACAGAAGCUTT-3’（SEQ ID NO：3）,
siRNA-Antisense strand：5’-AGCUUCUGUAUGGAACGGGTT-3’（SEQ ID NO：4）.
2. applications of the siRNA described in claim 1 in drug-resistant microorganism drug resistance is reduced.
3. application according to claim 2, it is characterised in that the drug-resistant microorganism is containing integrase gene and drug resistance
The drug-resistant microorganism of box gene.
4. application according to claim 3, it is characterised in that the drug-resistant microorganism is containing integrase gene and drug resistance
The antibiotic-resistance E. coli of box gene.
5. the application according to claim 3 or 4, it is characterised in that the integrase gene selected from intI1, intI2,
intI3。
6. the application according to claim 3 or 4, it is characterised in that the drug resistant gene box selected from aad, aac, dfr,
bla、oxa、cat、ere、sat、grr、str。
7. applications of the siRNA described in claim 1 in overriding resistance microbial reagent is prepared.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610961933.6A CN106566830A (en) | 2016-11-04 | 2016-11-04 | SiRNA for regulating expression of integrase and drug-resistant gene cassette and application thereof |
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| Publication Number | Publication Date |
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| CN106566830A true CN106566830A (en) | 2017-04-19 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154291A (en) * | 2011-01-13 | 2011-08-17 | 华南理工大学 | SiRNA sequence for inhibiting integration of antibiotic resistance gene cassette |
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2016
- 2016-11-04 CN CN201610961933.6A patent/CN106566830A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154291A (en) * | 2011-01-13 | 2011-08-17 | 华南理工大学 | SiRNA sequence for inhibiting integration of antibiotic resistance gene cassette |
Non-Patent Citations (1)
| Title |
|---|
| 鲁玉侠: "食源微生物耐药基因水平传播抑制研究", 《中国博士学位论文全文数据库 基础科技辑》 * |
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