CN109679959A - A kind of heterozygosis antisense oligonucleotides and its application with Mce4a gene complementation - Google Patents
A kind of heterozygosis antisense oligonucleotides and its application with Mce4a gene complementation Download PDFInfo
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Abstract
The application provides a kind of and Mce4a gene complementation heterozygosis antisense oligonucleotides, the nucleotide sequence of the heterozygosis antisense oligonucleotides is as shown in SEQ ID NO:1, it is very low that its base modification mode is the 1st to the 5th bit base, the 15th to the 19th bit base LNA modifies its undershooting-effect, and thermodynamic stability is very good, it is not easy to be degraded in environment in vivo, with the help of no transfection reagent, mycobacterium tuberculosis silencing agent can may be used as by cell automatic absorption.
Description
Technical field
The application belongs to field of biotechnology, in particular to a kind of heterozygosis antisense oligonucleotides with Mce4a gene complementation
And its application.
Background technique
Tuberculosis of spine is secondary disease, and protopathy is pulmonary tuberculosis, alimentary canal tuberculosis or scrofula etc., is made through blood circulation approach
At tuberculosis of bone and joint.
The treatment of tuberculosis of spine still suffers from disease incidence height, the increase of refractory case, again operability height etc. problem.It is existing
Some treats the conventional medicine molecule that chemotherapeutics lungy is non-genomic, non-nano, non-targeted molecule, in addition spine node
The barrier action of nuclear hardening densification lesion wall, causes drug molecule to be difficult to enter into lesion tissue.Many studies have shown that nanometers are micro-
Grain is easy to break through barrier, into target tissue, target cell, to improve the therapeutic effect of drug and mitigate the poison of chemotherapeutics
Side effect, so we determine the newest research results using the building of nano target Gene Microparticles, to develop antituberculosis therapy
New drug, novel form, new administration mode, the treatment for anti-consumptive disease failure tuberculosis of spine search out a kind of effective method.It grinds
The albumen for studying carefully discovery Mce4a gene coding can cause the inflammatory reaction of body, and during the immune response of host
It plays an important role.Research shows that the expression of Mce4a gene in smegmatis mycobacterium can be made to reduce using silent technology, from
And the infection virulence of smegmatis mycobacterium is caused to decline.Since smegmatis mycobacterium and mycobacterium tuberculosis have in gene expression
There is the homology of height, therefore can be considered and treat potential target gene for the Mce4a gene in mycobacterium tuberculosis as silencing.
And the research of mycobacterium tuberculosis silencing effect is had shown that, it can phase on effective reticence knot mycobacterium tuberculosis using antisense technology
Target gene is closed, the silencing effect to mycobacterium tuberculosis is reached.
Antisense technology refers to by artificial synthesized or naturally occurring oligonucleotides in the way of base pair complementarity, with
Target gene mRNA complementation combines, and so that the mRNA of target gene is degraded by various mechanism or it is inhibited to be translated as coding albumen, from
And inhibit the expression of target gene.In gene silencing effect, this antisense technology can be for positioned at Mycobacterium tuberculosis cell
In Mce4a gene come design synthesis, Mce4a gene plays important in course of infection of the mycobacterium tuberculosis to host cell
Effect, i.e., during the transcription of mycobacterium tuberculosis, expression have important regulating and controlling effect.In mammalian cells,
When the Mce4a gene in the mycobacterium tuberculosis of infection cell mutates, then the decreased growth of mycobacterium tuberculosis or dead
It dies.
Summary of the invention
The application is the research to tuberculosis of spine cryptiogene, research using the Mce4a gene in mycobacterium tuberculosis as
Target gene, according to the design principle of antisense sequences, specifically, antisense sequences length nucleic acid need to be suitable for sufficiently avoiding in target sequence
The formation of special RNA secondary structure avoids the presence of a large amount of unmethylated GC sequences in antisense sequences, avoids special in antisense sequences
The formation of different fourtier structure, avoids the appearance that can increase or decrease the active specific base sequence of antisense sequences, and Preliminary design goes out
Candidate antisense sequences corresponding with Mce4a gene specific, as a result as shown in table 1, table 2, and according to the specific design of antisense sequences
Principle (table 3, table 4 for done on the basis of 2 result of table further analysis) from table 1, table 2 selects suitable antisense sequence
Column.Since the antisense sequences designed are negatively charged, autonomous can not enter in mycobacterium tuberculosis, and even if
It enters in mycobacterium tuberculosis and also can rapidly be degraded by RNase III, therefore needs to the above-mentioned antisense selected
Sequence carries out lock nucleic acid modification.By design synthesize it is corresponding with Mce4a gene specific, by oligonucleotides-deoxyribose core
Acid-oligonucleotides composition heterozygosis antisense oligonucleotides (LNA-DNA-LNA Gapmer antisense) sequence, and detect it
Influence to Mce4a gene expression in mycobacterium tuberculosis, finishing screen selects one kind can be with Mce4a base in mycobacterium tuberculosis
Because of the special complementary heterozygosis antisense oligonucleotides combined and there is silencing effect to the growth and breeding of mycobacterium tuberculosis.
Specifically, according to antisense sequences design principle, firstly, for all possible antisense sequences carried out G/C content,
Oligonucleotides combines energy (oligo binding energy) and whether there is or not the detections of GGGG sequence.In view of above-mentioned antisense sequences
Design principle, therefore -7.5kcal/mol and the possibility antisense sequences without GGGG sequence can be less than by picking the free combination of oligo,
The results are shown in Table 1.
The gene order that table 1. is synthesized according to the design of oligo Conjugated free energy
Wherein 441~459 position, 442~460 positions, 443~461 positions, 892~910 positions, 440~458 positions,
Contain ACTG, TAA, CCGG in the possibility antisense sequences of 445~463 positions and 891~909 positions, antisense sequences can be reduced
Activity, so eliminating these candidate sequences.Due to GEM 132 G/C content should in 40%~60%, 2~20 position,
The possibility antisense sequences needs of 3~21 positions, 632~650 positions, 894~912 positions and 895~913 positions remove.More than
Analysis carries out screening possible antisense sequences, only 1 it is found that if according on the basis of oligo Conjugated free energy and G/C content is considered
The antisense sequences of~19 positions: GGAACGGGACGACTTCTGA is most possible Mce4a gene specific antisense sequences.
Further, the design of antisense sequences also needs to consider its combination in addition to needing to consider above-mentioned oligo Conjugated free energy
The failure energy at position.Because antisense sequences can make the structure of target sequence change after being integrated to its target sequence, if its
The failure energy of binding site is too small, then can make the structure of target sequence that can not change, this means that antisense sequences cannot
Play the effect for inhibiting expression of target gene.Therefore, the application to the coded sequence (coding sequence, CDS) of Mce4a with
The value of the failure energy of the binding site of possible antisense sequences is calculated.Simultaneously to specific target sequence and other site sequences
Not pairs of possibility is calculated, and not pairs of likelihood value shows that more greatly target sequence forms RNA secondary structure in privileged site
Possibility is smaller, i.e., the antisense sequences the easy to be in connection, and design result is as shown in table 2.
The gene order that table 2. is synthesized according to the design of the failure energy (Δ G disruption) of binding site
In group sequence shown in table 2,435~453 positions, 441~459 positions, 442~460 positions, 639~657
It sets, 642~660 positions, the sequence of 643~661 positions, which contains, reduces the active ACTG of antisense sequences, TAA, CCGG sequence, therefore
Remove these possible antisense sequences from candidate sequence.Remove the destruction of binding site in remaining candidate antisense sequences again
Energy (Δ G disruption) value is less than -10 antisense sequences, and therefore, residue has 6 candidate antisense sequences to meet the requirements, such as
Shown in table 3.
Failure energy (the Δ G disruption) value of 3. binding site of table is greater than -10 candidate sequence
Applicants have discovered that 590~608 positions, two intervals GG of the sequence of 591~609 positions are only in above-mentioned sequence
There are a base (base), fourtier structure (Tetraplexes) easy to form.Therefore, the two candidate sequences are excluded, are obtained most
Possible Mce4a specific antisense sequence, as shown in table 4.
Table 4. is most possibly failure energy (Δ G disruption) value of Mce4a specific antisense sequence nucleotide sequence
Applicants have discovered that the sequence of 1~19 position meet simultaneously oligo Conjugated free energy calculate and binding site it is broken
Bad energy (Δ G disruption) value calculates.
Therefore, the application selects the candidate sequence of 1~19 position as Mce4a gene specific antisense sequences, such as SEQ ID
Shown in NO:1, specifically: GGAACGGGACGACTTCTGA, the gene order are distributed in 5 ' noncoding regions, code area and 3 ' non-volumes
Code area, the length is 10-30 bases, specially 19 bases.
Since the group sequence has negative electrical charge, which can not independently enter mycobacterium tuberculosis and work as
In, moreover, can rapidly be degraded by RNase III entering in mycobacterium tuberculosis, therefore, the application for
Above-mentioned Mce4a specific antisense sequence is modified, and particularly, is modified using lock nucleic acid.Lock nucleic acid (locked nucleic
Acid, LNA) it is a kind of novel oligonucleotide derivative, 2 '-O of B-D- ribofuranose in structure, 4 '-C are acted on by shrink
Form Oxymethylene bridge, sulphur methylene bridge or the amine methylene bridge of annular, the structure latches of furanose C3. inner mold N configuration,
Form the condensation structure of rigidity.LNA as a kind of new antisense nucleic acid, have with DNA/RNA it is powerful hybridize affinity, antisense
The advantages that activity, nuclease-resistant ability, good water solubility and internal nontoxicity.That is, antisense oligonucleotides provided by the present application is to repair
Heterozygosis antisense oligonucleotides after decorations, for nucleotide sequence as shown in SEQ ID NO:1, base modification mode is the 1st to the 5th
Base, the 15th to the 19th bit base LNA modification, specifically:
5 ' -+G+G+A+A+CGGGACGACT+T+C+T+G+A-3 ' (+indicate the part LNA).
Applicants have discovered that heterozygosis antisense oligonucleotides undershooting-effect provided by the present application is very low, and Thermodynamically stable
Property is very good, it is not easy to it is degraded in environment in vivo, it, can be by cell automatic absorption with the help of no transfection reagent.
Heterozygosis antisense oligonucleotides provided by the present application may be used as mycobacterium tuberculosis silencing agent.
Heterozygosis antisense oligonucleotides provided by the present application can be also used for preparing mycobacterium tuberculosis silencing agent.
The application also provides a kind of mycobacterium tuberculosis silencing agent, and the silencing agent includes that foregoing heterozygosis antisense is few
Nucleotide.
Further, the dosage form of the silencing agent includes oral agents, external preparation, freeze dried powder and injection, including tablet,
Other dosage forms such as capsule, granule, dripping pill, powder, pill, paste, freeze dried powder and injection.
Detailed description of the invention
Fig. 1 is the Ct value schematic diagram that each group is measured by real-time quantitative PCR reaction in embodiment.
Specific embodiment
Illustrate the scheme of the application by the following examples.
(1) research object and grouping
This experimental study is divided into seven groups, and every group includes three culture bottles: A group (number 1-3) is not do any processing
Blank control group；It is 0.1 μM, 1 μM, 10 μM that B group, C group, D group (number is respectively 4-6,7-9,10-12), which are respectively by concentration,
Antisense TYE563-Mce4a DNA processing negative control group；(number is respectively 13-15,16-18,19- for E group, F group, G group
It 21) be respectively by concentration is, 0.1 μM, 1 μM, the experimental group that handles of 10 μM of antisense TYE563-Mce4a LNA/DNA/LNA.This
Experimental study is audited by Ningxia Medical University's hospital general's Institutional Review Board to be ratified.
(2) key instrument and reagent
1. key instrument: ultracentrifuge (Hitachi, Japan), ultrasonic oscillator (Branson company, the U.S.), CO2
Cell incubator (Fisher company, the U.S.), 721 type spectrophotometers (Chinese Shanghai You Ke company), NanoDrop Lite points
Light photometer (Fisher Scientific company, the U.S.), MPFastprep24 sample homogeneous crushing system (U.S. MP
Biomedicals company), Premier Primer 5.0 (Premier Biosoft company, the U.S.), real-time PCR (beauty
ALT company, state), Icycler fluorescence spectrum thermal cycler (Bio-Rad company, the U.S.).
2. main agents: antisense TYE563-Mce4a LNA/DNA/LNA (Exiqon company, the U.S.),
Middlebrooke7H9 fluid nutrient medium (Sigma-aldrich company, the U.S.), the extracts kit (BeiJing, China hundred of total serum IgE
Tyke Bioisystech Co., Ltd), s μ perscript III reverse transcriptase (Invitrogen company, the U.S.), RT2SYBR
Green qPCR artificial mixture (archaeal dna polymerase including thermal starting, dNTPs, Mg2+With fluorescent dye SYBR Green I)
(German Qiagen company), Mce4a special primer (biotech firm, New England, the U.S.).
(3) experimental method
1. the design and synthesis of heterozygosis antisense oligonucleotides
Due to itself negatively charged characteristic of traditional antisense oligonucleotides siRNA, tuberculosis generally can not be independently entered
It in mycobacteria, and is to make it into mycobacterium tuberculosis also be degraded by RNaseIII rapidly.In order to overcome this
A little sciences problems, the application have carried out lock nucleic acid modification to designed Mce4a gene specific Antisensedigonucleotsequence sequence out.Lock
Nucleic acid (locked n μ ncleic acid, LNA) is a kind of novel oligonucleotide derivative, B-D- ribofuranose in structure
2'-O, 4'-C act on Oxymethylene bridge, sulphur methylene bridge or the amine methylene bridge to form annular, the knot of furanose by shrink
Structure is locked in the N configuration of C3' inner mold, forms rigid condensation structure.And LNA is as a kind of new antisense nucleic acid, have with
DNA/RNA powerful hybridization affinity, antisense activity, nuclease-resistant ability, good water solubility and it is non-toxic in vivo the advantages that.
According to the design principle of antisense LNA/DNA/LNA Gapmer, Mce4a gene specific antisense TYE563- is determined
The sequence of Mce4a LNA/DNA/LNA (LNA/DNA/LNA Gapmer antisense), information are as follows:+G+G+A+A+
CGGGACGACT+T+C+T+G+A (+indicate the part LNA).Due to antisense LNA-DNA-LNA Gapmer itself particularity and
The reason of U.S.'s intellectual property protection method；Only have a biotech firm (Exiqon company, the U.S.) that can close in the world at present in addition
Such structure of the synthesis of method, so having subscribed above-mentioned heterozygosis antisense oligonucleotides acid fragment from the said firm.It is special in order to facilitate it is detected
Property and the observation to experimental result, to the 5 of heterozygosis Antisensedigonucleotsequence sequence, ' end has carried out the modification of TYE563 fluorescence.Instead
Adopted TYE563-Mce4a LNA/DNA/LNA:5 '-(TYE563)+G+G+A+A+CGGGACGACT+T+C+T+G+A-3 ', antisense
TYE563-Mce4a DNA/DNA/DNA:5 '-(TYE563) GGAACGGGACGACTTCTGA-3 ', uses Invitrogen company
Antisense TYE563-Mce4a LNA/DNA/LNA and antisense TYE563-Mce4a DNA/DNA/DNA are carried out respectively without enzyme water
Dissolution.
2. the Multiplying culture of mycobacterium tuberculosis (H37Ra)
Mycobacterium tuberculosis (H37Ra) is from American Type Culture collection warehousing (American Type C μ lt μ re
Collection, ATCC) purchase.Mycobacterium tuberculosis is inoculated into Middlebrooke7H9 fluid nutrient medium and is cultivated.
The ACD anti-coagulants and 0.4% glycerine of addition 10% into Middlebrooke7H9 fluid nutrient medium, and in culture bottle
Mycobacterium tuberculosis every 24 hours carry out one time 20 seconds or so micro-ultrasonic concussion, to prevent bacterium conglomerate.It will knot
Core mycobacteria is placed in temperature and is 37 DEG C, is 5%CO containing volume fraction2It is cultivated in the cell incubator of humidified air, with
Keep the stabilization of inoculum pH value.Always culture to mid-log phase (optical density at 600nm [OD600]=
It 0.5), is all mycobacterium tuberculosis (H37Ra) of the culture to mid-log phase used in all following experiments.
3. processing of the heterozygosis antisense oligonucleotides to mycobacterium tuberculosis
The above-mentioned culture of 10ml is separately added into 21 bacteria culture bottles to the mycobacterium tuberculosis of mid-log phase, wherein A
Group (number 1-3) does not do any processing；B group (number 4-6), C group (number 7-9), D group (number 10-12) are respectively
Be added concentration be 0.1 μM, 1 μM, 10 μM of antisense TYE563-Mce4a DNA/DNA/DNA handled；E group (number 13-
15), F group (number 16-18), G group (number 19-21) be separately added into concentration be 0.1 μM, 1 μM, 10 μM of antisense
TYE563-Mce4a LNA/DNA/LNA is handled.Antisense TYE563-Mce4a DNA/DNA/DNA and antisense TYE563-
Mce4a LNA/DNA/LNA carries out processing in 32 hours by a definite date to the mycobacterium tuberculosis of above-mentioned grouping respectively；Later again with
The enhancing that same concentration carries out mycobacterium tuberculosis 32 hours is handled；Later again with same concentration to tuberculosis branch
Bacillus carries out enhancing processing in 32 hours by a definite date.The micro-ultrasonic for carrying out 25 seconds or so to culture bottle daily during drug-treated shakes
It swings, to prevent bacterium conglomerate.
4. the extraction and quality inspection of total serum IgE after the processing of heterozygosis antisense oligonucleotides
Before real-time quantitative PCR reaction, the total RNA content in the mycobacterium tuberculosis after need to being subject to processing to each group is carried out
Detection, it is therefore an objective to the purity Yu content of total serum IgE in each group bacterium be made to reach the requirement of experiment of A260/A280≤1.8.It is anti-to heterozygosis
After oligonucleotide is handled mycobacterium tuberculosis 96 hours, under the conditions of 4000g, 10 points are centrifuged to the mycobacterium tuberculosis of culture
Clock removes supernatant；Later, 1ml extraction agent is added into centrifuge tube makes precipitating suspend again；Then suspension is moved into shake
Pipe is swung, and the crushing of mycobacterium tuberculosis uses MPFastprep24 sample homogeneous crushing system, illustrates according to producer to tuberculosis
The concussion that mycobacteria carries out special program crushes, while generating superheating phenomenon in crushing process in order to prevent, therefore uses dry ice
It is cooled down frequently.After being observed visually mycobacterium tuberculosis and thoroughly being crushed, under the conditions of 12000g, it is centrifuged 5 minutes, and
Supernatant is moved into 2ml Eppendorf pipe.Then, be added 200 μ l without enzyme chloroform, high speed concussion 20 seconds；Exist later
Be centrifuged 15 minutes under the conditions of 13000g, gained supernatant is moved into rapidly in 1.5ml Eppendorf pipe after centrifugation, and be added etc.
(about 350 μ l) of amount is without enzyme isopropanol；It stands after ten minutes, again under the conditions of 13000g, is centrifuged 10 minutes at room temperature, and
Remove supernatant；The 70% of 800 μ l is added into centrifuge tube later makes precipitating suspend again without enzyme ethyl alcohol；Then in 13000g item
Under part, it is centrifuged 10 minutes, and remove supernatant.Finally after ultra-clean workbench is 8 minutes dry, dissolving without enzyme water for 30 μ l is added
It is precipitated in centrifuge tube.The purity and content of total serum IgE are measured using NanoDrop Lite spectrophotometer.
The design and synthesis of 5.Mce4a primer
When designing Mce4a special primer, 3 ' the mRNA segments pair that may be generated after Mce4a Gene degradation in order to prevent
The interference that experimental result generates, therefore the appearance of antisense action site is specially avoided in design primer.This experiment primer is set
Meter is carried out using 5.0 software of Premier Primer, wherein using 16s rRNA as internal reference.Primer entrusts DNA synthesis science and technology public
Department is designed and synthesizes, and obtained primer dissolve using no enzyme water and ultimate density is tuned into 100 μM, following experiments
In the concentration of Mce4a primer used be 0.5 μM.
6. Real-time PCR Analysis
It is carried out according to total RNA extraction reagent box specification, extracts every group of above-mentioned each group and be subject to processing tuberculosis branch containing three parts
The total serum IgE of bacillus, reverse transcription are single-stranded cDNA.Mce4a primer sequence are as follows: Mce4a, positive-sense strand: 5'-
GAGTAGTCGGAGGTCACT-3', antisense strand: 5'-CATCGGTCTGTCTAATAACG-3'；16s rRNA, positive-sense strand: 5'-
TCCCGGGCCTTGTACACA-3', antisense strand: 5'-CCACTGGCTTCGGGTGTTA-3'.Real-time quantitative polymerase chain reaction
Using two-step method, reaction condition is as follows: 95 DEG C 3 minutes, 1 circulation；95 DEG C 10 seconds, 60 DEG C 30 seconds, repeat 40 circulation；55
DEG C -95 DEG C, 0.5 DEG C of increment, 10 seconds, 1 circulation.
Real-time quantitative analysis uses the Icycler fluorescence spectrum thermal cycler of Bio-rad company, and the measurement of Mce4a uses
The method of Ct value relative quantification, and 16s rRNA is used to be corrected as internal reference.
(4) statistical method
Data analysis is carried out using SPSS19.0 (SPSS, the U.S.) statistical software, measurement data is indicated using `x ± s；It is more
Comparison among groups use one-way analysis of variance, compare two-by-two between group and are examined using LSD-t, and inspection level α is 0.05.With P ﹤ 0.05
Think that difference has statistical significance.
(5) interpretation of result
1. total serum IgE quality inspection result
After processing, the purity and content of total serum IgE use NanoDrop Lite spectrophotometric to each group mycobacterium tuberculosis
Meter is determined, after measured in extracted each group mycobacterium tuberculosis the purity Yu content of total serum IgE be A260/A280≤
1.8, the requirement for having reached experiment is as shown in table 5.
The purity and content analysis of the total serum IgE extracted in 5. each group mycobacterium tuberculosis of table
2. real-time quantitative PCR result
The relative expression quantity result of 2.1 Δ Δ Ct methods calculating each group Mce4a gene
It is reacted for real-time quantitative PCR, using in Icycler fluorescence spectrum thermal cycler measurement each group mycobacterium tuberculosis
The Ct value of Mce4a gene.The analysis of each group Mce4a gene relative expression quantity is calculated using Δ Δ Ct method, and detailed step is such as
Under: the first step, reference gene uniform differences between samples (Ct=Ct target gene-Ct reference gene)；Second step, other samples and
Check sample compares (Ct=Ct handles sample-Ct check sample)；Finally, being calculated (multiple variation=2 using formula-△△Ct).2 between seven groups of statistical analysis A, B, C, D, E, F, G-△△CtThere are statistical significance (F=24.826, P ﹤ for the difference of value
0.05)；And when being compared two-by-two between tetra- groups of A, B, C, D, 2-△△CtStatistical significance is all not present in the difference of value, is P > 0.05；
E, in tri- groups of F, G any one group respectively compared with remaining is six groups when, 2-△△CtThere is statistical significance in the difference of value, be P ﹤
0.05；E, 2 between tri- groups of F, G-△△CtThere are statistical significances for the difference of value, are embodied in E group (0.7541 ± 0.1324)
When compared with F group (0.5517 ± 0.0594), P ﹤ 0.05；E group (0.7541 ± 0.1324) and G group (0.2316 ± 0.1573) phase
Than when, P ﹤ 0.05；When F group (0.5517 ± 0.0594) is compared with G group (0.2316 ± 0.1573), P ﹤ 0.05 (table 6).
Influence of the 6. various concentration heterozygosis antisense oligonucleotides of table to Mce4a gene expression in mycobacterium tuberculosis
Note: PEF﹤ 0.05, PEG﹤ 0.05, PFG﹤ 0.05
Influence of the 2.2 heterozygosis antisense oligonucleotides to Mce4a gene expression in each group mycobacterium tuberculosis
By the above-mentioned each group Ct value measured by real-time quantitative PCR reaction, 6.0 software of Graphpad Prism is used
It is calculated, and processing is patterned to calculated result, as a result as shown in Figure 1.It can be seen from the figure that blank control
Group A group (1.0071 ± 0.1468) and negative control group B group (0.9541 ± 0.1020), C group (0.9857 ± 0.1072), D group
The expression of Mce4a gene, which is not affected by, in (0.9275 ± 0.0902) mycobacterium tuberculosis significantly affects；And work as mycobacterium tuberculosis
When being handled respectively by the heterozygosis antisense oligonucleotides of various concentration, the expression of Mce4a gene is lowered in mycobacterium tuberculosis, specifically
Show as when the concentration of heterozygosis antisense oligonucleotides is 0.1 μM, the relative expression quantity of Mce4a gene be (0.7541 ±
0.1324)；When the concentration of heterozygosis antisense oligonucleotides is 1 μM, the relative expression quantity of Mce4a gene be (0.5517 ±
0.0594)；When the concentration of heterozygosis antisense oligonucleotides is 10 μM, the relative expression quantity of Mce4a gene be (0.2316 ±
0.1573).The concentration that real-time quantitative PCR result shows as heterozygosis antisense oligonucleotides is higher, Mce4a base in mycobacterium tuberculosis
The expression of cause is lower.
Heterozygosis antisense widow's core of Mce4a specific oligonucleotide acid-DNA-oligonucleotides composition provided by the present application
Thuja acid meets simultaneously: (1) heterozygosis antisense oligonucleotides autonomous can enter in mycobacterium tuberculosis；(2) heterozygosis antisense is few
Nucleotide can be completed by prokaryotic cell RNase H mechanism present in Mce4a gene expression in mycobacterium tuberculosis
Inhibition.
Above-mentioned experimental result shows, the expression of Mce4a gene in blank control group and negative control group mycobacterium tuberculosis
It is not affected by apparent influence, illustrates that the antisense TYE563-Mce4a DNA/DNA/DNA of various concentration will not influence tuberculosis branch bar
The expression of Mce4a gene in bacterium；The expression of Mce4a gene is significantly affected in each experimental group mycobacterium tuberculosis, is illustrated not
It will affect the expression of Mce4a gene in mycobacterium tuberculosis with the antisense TYE563-Mce4a LNA/DNA/LNA of concentration, show
Higher for heterozygosis antisense oligonucleotides acid concentration, the expression of Mce4a gene is lower in mycobacterium tuberculosis.For the biography of tuberculosis of spine
System treatment method includes drug therapy and operative treatment, since TB focus hardens the presence of appearance and " barrier " effect of wall,
So that drug therapy and operative treatment is ineffective, there is an urgent need to develop new antituberculosis therapy drug, dosage form with to prescription
Formula, to effectively kill the mycobacterium tuberculosis in lesion tissue.In conjunction with this experimental studies results it is found that Mce4a gene is special
Different heterozygosis antisense oligonucleotides can effectively inhibit the expression of Mce4a gene in mycobacterium tuberculosis.In addition heterozygosis
The design of antisense oligonucleotides is carried out in strict accordance with basic principle, has accomplished that length nucleic acid is suitable for, and few in design heterozygosis antisense
Special secondary structure, GC sequence, the fourtier structure of not methylating largely are substantially avoided when nucleotide and can increase or decrease this
The appearance of the problems such as sequence of nucleotide activity.Also as described above, gene nano Target Particles technology be can yet be regarded as a kind for the treatment of
The good way of tuberculosis of spine.
In conclusion heterozygosis antisense oligonucleotides gene molecule provided by the present application sinks as what tuberculosis of spine silencing was treated
Silent gene.
Combine detailed description and exemplary example that the application is described in detail above, but these explanations are simultaneously
It should not be understood as the limitation to the application.It will be appreciated by those skilled in the art that without departing from the application spirit and scope,
A variety of equivalent substitution, modification or improvements can be carried out to technical scheme and embodiments thereof, these each fall within the application
In the range of.The protection scope of the application is determined by the appended claims.
Sequence table
<110>hospital general, Ningxia Medical University
<120>a kind of heterozygosis antisense oligonucleotides and its application with Mce4a gene complementation
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
ggaacgggac gacttctga 19
Claims (8)
1. a kind of and Mce4a gene complementation heterozygosis antisense oligonucleotides, which is characterized in that it is distributed in 5 ' noncoding regions, compiles
Code area and 3 ' noncoding regions, the length is 10-30 bases.
2. heterozygosis antisense oligonucleotides according to claim 1, which is characterized in that its nucleotide sequence such as SEQ ID NO:
Shown in 1, base modification mode is the 1st to the 5th bit base, the 15th to the 19th bit base LNA modification.
3. heterozygosis antisense oligonucleotides according to claim 1, which is characterized in that the heterozygosis antisense oligonucleotides processization
Learn modification.
4. heterozygosis antisense oligonucleotides according to claim 3, which is characterized in that its chemical modification is lock nucleic acid.
5. the purposes that any one of Claims 1-4 heterozygosis antisense oligonucleotides is used as the agent of mycobacterium tuberculosis silencing.
6. the purposes that any one of Claims 1-4 heterozygosis antisense oligonucleotides is used to prepare mycobacterium tuberculosis silencing agent.
7. a kind of mycobacterium tuberculosis silencing agent, which is characterized in that the silencing agent includes described in any one of Claims 1-4
Heterozygosis antisense oligonucleotides.
8. silencing agent according to claim 7, which is characterized in that the dosage form of the silencing agent include oral agents, external preparation,
Freeze dried powder and injection, including tablet, capsule, granule, dripping pill, powder, pill, paste, freeze dried powder and injection
Deng other dosage forms.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317834A (en) * | 2019-04-29 | 2019-10-11 | 宁夏医科大学总医院 | A method of synthesis pRNA-3WJ-siLNAgapmer-aptamer |
-
2019
- 2019-01-31 CN CN201910097713.7A patent/CN109679959A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| AMITA CHANDOLIA等: "Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach", 《MICROBIOLOGICAL RESEARCH》 * |
| ERIN K SULLY等: "Antisense antimicrobial therapeutics", 《CURRENT OPINION IN MICROBIOLOGY》 * |
| REMO GEORGE等: "Use of siRNA molecular beacons to detect and attenuate mycobacterial infection in macrophages", 《WORLD J EXP MED》 * |
| 佚名: "登录号：F5BH98", 《UNIPROT》 * |
| 吴姝 等: "结核分枝杆菌Mce4蛋白结构与功能的生物信息学分析", 《中国病原生物学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317834A (en) * | 2019-04-29 | 2019-10-11 | 宁夏医科大学总医院 | A method of synthesis pRNA-3WJ-siLNAgapmer-aptamer |
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