CN101463351A - External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation - Google Patents
External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation Download PDFInfo
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- CN101463351A CN101463351A CNA2009100365955A CN200910036595A CN101463351A CN 101463351 A CN101463351 A CN 101463351A CN A2009100365955 A CNA2009100365955 A CN A2009100365955A CN 200910036595 A CN200910036595 A CN 200910036595A CN 101463351 A CN101463351 A CN 101463351A
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Abstract
The invention discloses an EGS of 12nt, which can conduct RNase P ribozyme and comprises EGS which is based on RNA and has the nucleotide sequence to be shown in SEQ ID NO: 1, and EGS that is based on DNA and has the nucleotide sequence to be shown in SEQ ID NO: 2. The EGS can effectively inhibit the expression of HCMV UL49 gene. The EGS with the characteristic of DNA has the silent gene efficiency of 99%, and the EGS with the characteristic of RNA has the silent gene efficiency of 98%. Compared with the existing EGS which can cause the UL49 gene to be silent, the EGS of the invention has the sequence which only contains 12 basic groups, thus not only having the shortest nucleic acid molecules, but also effectively and specially leading the expression of the UL49 gene to be silent. The EGS of the invention is used for researching and developing the anti-HCMV drug, and can greatly reduce the cost when the drug is researched and applied.
Description
Technical field
The invention belongs to molecular biology and medical field, be specifically related to a kind of external guide sequence and application in the anti-HCMV medicine of preparation thereof of guiding RNase P ribozyme.
Background technology
RNase P extensively is present in various intracellular a kind of nucleoprotein complex bodys, it can be specific in vivo 5 ' end of shearing tRNA precursor (ptRNA), form sophisticated tRNA.
RNase P has identification substrate secondary structure but not the characteristics of identification substrate nucleotide sequence, and its pattern substrate will comprise two important documents at least: 1, free CCA-3 ' end; 2, special complementary double-stranded region.Therefore as long as two RNA molecules form the mixture that is similar to the ptRNA molecular structure, can be discerned and cut by RNase P.
Can guiding RNase P the RNA molecule of cleavage site on the identification said target mrna be called as external guide sequence (External Guide Sequence, EGS).In theory,, use potential cleavage site complementation on suitable EGS and the said target mrna so, can carry out the specificity cutting to said target mrna in the mixture by guiding RNase P ribozyme as long as have RNase P potential cleavage site on the said target mrna.EGS can be divided into based on RNA EGS (RNA-based EGS) and based on the EGS (DNA-based EGS) of DNA, these two kinds of EGS can both effectively guide cutting substrate mRNA.
The human cytomegalovirus (human cytomegalovirus, HCMV) be a kind of in the different ethnic populations in the whole world wide-scale distribution and the very high pathogenic agent of infection rate.Discovering that at present it is to cause transplant infection, intrauterine infection that HCMV infects the main harm that causes, and causes fetal abortion, osteomyelodysplasia, deformity, mental retardation etc., therefore is the key subjects in medical science and orthogenics and the bone marrow transplantation research.
The emphasis of present anti-HCMV medicament research and development is to seek the medicine more efficient, that resistance is littler.Find in the culture experiment that outside virosome disappearance UL49 gene pairs HCMV is lethal, so the UL49 gene is expected to one of target gene that becomes anti-HCMV medicament research and development.
The inventor has manually designed several external guide sequence EGS molecules, these EGS molecular energies and substrate (UL49 gene) mRNA forms the nucleotide sequence of similar ptRNA, and can substrate (UL49 gene) mRNA be cut guiding RNase P, thereby blocked the UL49 expression of gene.These EGS length are all about 54 bases, these EGS molecule base length are all long, though can reticent effectively specifically UL49 expression of gene, but need very high cost during as medicament research and development and application, thereby for researching and developing and using and bring bigger obstacle, so be necessary to seek the medicament research and development that the shorter EGS molecule of a kind of length is used for anti-HCMV.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide the efficient high specific of a kind of bootable RNase P ribozyme to get reticent HCMV virus UL49 genetic expression, and length only has the external guide sequence (EGS) of 12nt, comprises based on the EGS of RNA with based on the EGS of DNA.
Another object of the present invention is to provide the application of above-mentioned EGS in the anti-HCMV medicine of preparation.
Above-mentioned purpose of the present invention is achieved by following scheme:
The inventor is a target sequence with the mRNA fragment of Human cytomegalic inclusion disease virus (HCMV) ULM9 gene, at this sequences Design external guide sequence (EGS), obtain the EGS of the 12nt of a bootable RNase P ribozyme, comprise that its nucleotide sequence of EGS based on RNA is shown in SEQ ID NO:1, with the EGS based on DNA, its nucleotide sequence is shown in SEQ ID NO:2.
The inventor utilizes carrier cloning UL49 gene, and has made up the platform of the clone of stably express UL49 as checking EGS gene silencing efficient; Then the inventor will detect the gene silencing efficient of EGS then based on the EGS of RNA with based on the EGS of the DNA clone of the above-mentioned energy of transfection energy stably express UL49 respectively.
Detected result proves: though EGS of the present invention be RNA character or DNA character all the UL49 expression of gene is had high specific and restraining effect efficiently: its gene silencing efficient of the EGS of RNA character can reach 98%, RNA character EGS in use can preferred 0.5～10nM concentration, and most effective to the inhibition of UL49 gene when the concentration of RNA character EGS is 2.5nM; Its gene silencing efficient of the EGS of DNA character can reach 99%, the concentration that DNA character EGS in use can preferred 10～100nM, and most effective to the inhibition of UL49 gene when the concentration of DNA character EGS is 40nM.
The disappearance of UL49 gene is lethal to HCMV, and EGS of the present invention can efficiently suppress the UL49 expression of gene, therefore can be used for preparing the medicine of anti-HCMV.
Compared with prior art, the present invention has following beneficial effect:
1, EGS of the present invention can efficiently suppress HCMV UL49 expression of gene, and its gene silencing efficient of the EGS of DNA character is 99%.Its gene silencing efficient of the EGS of RNA character is 98%, compares with the EGS of the reticent UL49 gene of existing energy, and its sequence of EGS of the present invention only has 12 bases, and still nucleic acid molecule is the not shortest, and efficient, the special reticent UL49 expression of gene of energy;
2, EGS of the present invention can the cell guiding endogenous and the abundant RNase P ribozyme of content the mRNA of UL49 gene is carried out irreversible cutting, thereby suppress the UL49 expression of gene, therefore the EGS of the present invention medicine that is used to prepare anti-HCMV has efficient and the littler characteristics of resistance;
3, EGS of the present invention is used to prepare the research and development of anti-HCMV medicine, because of its length weak point has only 12nt, so can significantly reduce medicament research and development and the cost when using.
Description of drawings
Fig. 1 detects the restraining effect histogram of RNA character EGS to the UL49 gene mRNA for fluorescence quantitative RT-RCR;
Fig. 2 detects the restraining effect histogram of DNA character EGS to the UL49 gene mRNA for fluorescence quantitative RT-RCR;
Fig. 3 detects RNA character EGS to the inhibition of UL49 expression of gene protein electrophorogram as a result for Western blot;
Fig. 4 detects DNA character EGS to the inhibition of UL49 expression of gene protein electrophorogram as a result for Western blot;
Wherein, 1 is the transfection reagent control group, and 2 are EGS experimental group of the present invention, and 3 are the sudden change control group, and 4 is the nonsense control group, 5 positive control groups, and 6 is the Hela control group.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, but specific embodiment is not done any qualification to the present invention.
The acquisition of embodiment 1 EGS
1, search candidate's EGS molecule in the mRNA sequence of HCMV UL49 gene, its search principle is as follows:
A. in target gene (UL49 gene) mRNA sequence, search for UUCA or UUCG sequence;
B. the base of grappling cleavage site is G in 20～25 bases in UUCR sequence upstream;
C. the ring of candidate EGS molecule needs to be made up of 8～9 Nucleotide;
D. the Steml of candidate EGS molecule contains 1 G/C base pair at least, is preferably between 3～4;
E. contain 7～8 base pairs in the candidate EGS sequence at least;
F. the Steml of candidate EGS molecule and stem2 and target gene paired few nucleotide are preferably 5～6;
G. the Tm value of candidate EGS molecule is about 50 degree;
H. the binding site of candidate EGS molecule and target gene lacks secondary structure.
2, obtain the EGS molecule of one 12 base according to top 8 principles search, comprise the EGS of RNA character, its nucleotide sequence is shown in SEQ ID NO:1 and the EGS of DNA character, and its nucleotide sequence is shown in SEQ ID NO:2.This two sequences is sent to sequence Synesis Company, obtain the EGS nucleic acid fragment of RNA character and DNA character by businessman's chemosynthesis, synthetic simultaneously sudden change EGS and the nonsense EGS that is used to contrast, sudden change EGS is that 3 ' end at EGS of the present invention carries out point mutation the cleavage site of RNaseP is lost activity, and nonsense EGS is the EGS of the known array of another kind of virogene TK1.
The foundation of embodiment 2 checking EGS gene silencing efficient platforms
Present embodiment utilizes carrier cloning UL49 gene earlier, but make up the clone of stably express UL49 then, used carrier, clone, enzyme and all ingredients etc. are the commercially available prod among the embodiment, can select pcDNA3.1 (+)/myc plasmid as carrier, clone can be selected Hela clone, and double digestion can adopt BamHI and XhoI etc.
1, the acquisition of UL49 gene
Pcr amplification goes out the UL49 gene from the known array genome, as selecting pcr amplification UL49 gene from AD169 virus strain genome.
PCR reaction system cumulative volume is 100 μ l:LA Taq enzymes, 1 μ l, reaction buffer50 μ l, AD169 genomic dna template 5 μ l, UL49 upstream primer (nucleotide sequence is shown in SEQ ID NO:3) 5 μ l, UL49 downstream primer (nucleotide sequence is shown in SEQ ID NO:4) 5 μ l are supplemented to 100 μ l with sterile purified water.
PCR reaction conditions: first 95 ℃ of pre-treatment 3min, 95 ℃ of 30s then, 60 ℃ of 45s, 72 ℃ of 45s, totally 30 circulations, 72 ℃ of 5min again, 4 ℃ of 10min.
After agarose gel electrophoresis detected PCR result, gel reclaimed purifying UL49 gene fragment, and the operation reference reagent box specification sheets here gets final product.
2, make up the pcDNA3.1-UL49-myc plasmid
Inoculation is adopted commercially available plasmid extraction test kit after preserving the bacterial strain and enlarged culturing of pcDNA3.1 (+)/myc plasmid, according to test kit explanation extracting pcDNA3.1 (+)/myc plasmid.
Respectively above-mentioned pcDNA3.1 (+)/myc plasmid and UL49 gene are carried out BamHI and XhoI double digestion, the double digestion fragment that connects UL49 and pcDNA3.1 (+)/myc carrier then with the T4DNA ligase enzyme, then DNA being connected liquid is transformed in the DH5 α e. coli host bacteria, at Amp
+Substratum is chosen the clone, and identifies recon with PCR method and BamH I, Xho I double digestion, and the sequencing result explanation successfully makes up the pcDNA3.1-UL49-myc plasmid.Here related double digestion, the preparation of competent cell, T4 connect, connect liquid transformed into escherichia coli DH5 α and those skilled in the art's universal method in common knowledge is all adopted in tests such as PCR evaluation and double digestion evaluation.
3, the foundation of the HeLa clone of stably express UL49
Present embodiment is according to the Hela clone of pcDNA3.1 (+)/myc plasmid commodity operational guidance structure stably express UL49, as the platform of checking EGS gene silencing efficient.
A. growth conditions is good Hela cell transfer is in 24 orifice plates, and the DMEM culture medium culturing with the serum-free antibiotic-free begins to carry out transfection behind cell length to 50～80% in orifice plate;
B. get the above-mentioned pcDNA3.1-UL49-myc plasmid of 3 μ g, 12 μ l transfection reagent PlusReagent and 3 μ l liposome Lipofectamine
TMReagent all is dissolved in the 400 μ l serum-free antibiotic-free DMEM substratum and obtains mixed solution;
C. use the Hela cell in the serum-free antibiotic-free DMEM cleaning orifice, the amount with the every hole 400 μ l of above-mentioned mixed solution is added on the orifice plate then, and application of sample finishes 37 ℃ and handled 5 hours;
D. the flush away liposome adds perfect medium, continues to cultivate;
E. after the transfection 48 hours, add G418 (concentration of G418 is 800mg/ml) in above-mentioned substratum, select the cell enlarged culturing with anti-G418 after three weeks, the G418 that adds 200mg/ml in the substratum is as the usefulness of keeping the stably express cell cultures.
The reticent UL49 genetic expression of embodiment 3 EGS
The experimental group of present embodiment is: the Hela clone that embodiment 1 preparation gained EGS is transfected into the stably express UL49 gene of embodiment 2 structures.
The control group of present embodiment is: (1) sudden change control group: the sudden change EGS of embodiment 1 preparation according to the transfection conditions identical with experimental group, is transfected into the Hela clone of the stably express UL49 gene that embodiment 2 makes up; (2) nonsense control group: the nonsense EGS of embodiment 1 preparation according to the transfection conditions identical with experimental group, is transfected into the Hela clone of the stably express UL49 gene that embodiment 2 makes up; (3) the Hela clone of the stably express UL49 that positive controls: embodiment 2 makes up, but any EGS of untransfected; (4) transfection reagent control group: be meant the transfection conditions according to experimental group, the Hela clone of the stably express UL49 gene that embodiment 2 is made up is carried out transfection, but does not change any nucleic acid substances over to; (5) Hela control group: be meant the not simple Hela clone of oil-containing UL49 gene.
1. the Hela clone of the stably express UL49 that embodiment 2 is obtained is at 37 ℃, 5%CO
2Condition under place DMEM substratum (containing 100mg/ml penicillin, 100mg/ml Streptomycin sulphate and 200ml/mlG418) to cultivate;
2. transfection reagent adopts commercially available Lipofectamine2000, press the test kit operational guidance, the EGS that embodiment 1 is obtained with the final concentration be RNA character EGS be 2.5nM, that DNA character EGS is 40nM, the above-mentioned Hela cell of transfection, by flow cytometer transfection efficiency is detected, no matter be RNA character EGS or DNA character EGS, its transfection efficiency is all more than 97%;
3. fluorescence quantitative RT-RCR detects the restraining effect of EGS to the UL49 gene mRNA
After transfection EGS24 hour, collecting cell carries out the RNA extracting, and the RNA extracting can be adopted the TRizol method of Invitrogen company, and detailed operation by specification carries out.
Utilize Applied Biosystem ' s7300 system to carry out fluorescence quantitative RT-RCR and detect the restraining effect of EGS to the UL49 gene mRNA, concrete experimental procedure is as follows:
A. earlier according to the operation instructions of reverse transcription test kit, the extractive RNA of above-mentioned TRizol method is carried out reverse transcription obtain cDNA;
B. adopt the quantitative PCR kit (containing ROX) of the SYBR fluorescence dye of TAKARA company;
The nucleotide sequence of the upstream primer of UL49 gene is shown in SEQ ID NO:5;
The nucleotide sequence of the downstream primer of UL49 gene is shown in SEQ ID NO:6;
The nucleotide sequence of the upstream primer of confidential reference items β-actin is shown in SEQ ID NO:7;
The nucleotide sequence of the downstream primer of confidential reference items β-actin is shown in SEQ ID NO:8;
But the operation instructions in the equal reference reagent box of the reaction system of quantitative fluorescent PCR and reaction conditions.
Fluorescence quantitative RT-RCR detects the restraining effect of EGS to the UL49 gene mRNA, its result can find out by Fig. 1 and Fig. 2, except the mRNA level of Hela control group is zero, the mRNA level is minimum to be exactly EGS experimental group of the present invention, the mRNA level all reaches more than 3 times of experimental group in other control group, and the illustrative experiment group is to the restraining effect highly significant of UL49 gene.
In addition, the mRNA of UL49 gene descends 98% under the EGS of RNA character effect, the mRNA of UL49 gene descends 99% under the EGS of DNA character effect, is that the EGS or the EGS of DNA character of RNA character all has very high restraining effect to the mRNA of UL49 gene no matter this shows.
4.Western blot detects the restraining effect of EGS to the UL49 expression of gene protein
Collect the Hela cell as protein sample after transfection EGS24 hour, and wash protein sample, then protein sample and 4 * sample-loading buffer after the volume ratio mixing with 3:1, are boiled 5min in 100 ℃ of water and make protein denaturation with PBS.
Adopt the miniature vertical slab electrophoresis device of Bio-Rad company to prepare gel, by specification installs sheet glass, and prepares 12% separation gel and 5% successively and concentrate glue and make gel slab.Gel slab is put into behind the electrophoresis chamber above-mentioned protein sample point sample on gel slab, and 120V constant voltage leakage of electricity swimming finishes behind about 90min, then with reference to the operation instructions of molecular cloning guide this gel is changeed film, constant voltage 15V, electrotransfer 30min.
The film that shifts is sealed and antigen antibody reaction all can make reference to the text-book and the operation instructions of related kit is carried out, in the antigen antibody reaction, one anti-solution is anti-myc IgG of goat and the anti-Actin IgG of goat, all do the dilution of 1:500 (volume ratio) with confining liquid, the consumption of an anti-solution is at least 0.1ml/cm
2Membrane area, second antibody adopt the anti-goat IgG of horseradish peroxidase-labeled ox, and do the dilution of 1:4000 (volume ratio) with confining liquid, and two anti-consumptions also are 0.1ml/cm
2Membrane area.
According to this area common practice the result of above-mentioned antigen antibody reaction is carried out luminous development, as shown in Figure 3 and Figure 4, experimental group and all control groups all have band to show at Actin albumen place, and at UL49 albumen place, experimental group and Hela contrast do not have band, all the other control groups have band to show that the UL49 expression of gene has been suppressed fully in the illustrative experiment group.
In addition, the protein expression level of UL49 gene descends 99% under the effect of RNA character EGS, the protein expression level of UL49 gene descends 99% under DNA character EGS effect, is that the EGS or the EGS of DNA character of RNA character all has very high restraining effect to the protein expression of UL49 gene no matter this shows.
The external guide sequence of guiding RNase P ribozyme and the application sequence table in the anti-HCMV medicine of preparation thereof
SEQUENCE?LI?STING
＜110〉Ji'nan University
＜120〉external guide sequence of guiding RNase P ribozyme and the application in the anti-HCMV medicine of preparation thereof
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<170>PatentIn?version?3.5
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The external guide sequence of guiding RNase P ribozyme and the application sequence table in the anti-HCMV medicine of preparation thereof
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Claims (6)
1, the external guide sequence of guiding RNase P ribozyme, it is characterized in that this external guide sequence contains 12 bases, comprise external guide sequence based on RNA, its nucleotide sequence is shown in SEQID NO:1, with the external guide sequence based on DNA, its nucleotide sequence is shown in SEQ IDNO:2.
2, the application of the described external guide sequence of claim 1 in the anti-human cytomegalovirus's medicine of preparation.
3,, it is characterized in that described its working concentration of external guide sequence based on RNA is 0.5～10nM according to the described application of claim 2.
4,, it is characterized in that described its working concentration of external guide sequence based on DNA is 10～100nM according to the described application of claim 2.
5,, it is characterized in that its working concentration of external guide sequence based on RNA is 2.5nM according to the described application of claim 3.
6,, it is characterized in that its working concentration of external guide sequence based on DNA is 40nM according to the described application of claim 4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101634047A (en) * | 2009-08-12 | 2010-01-27 | 广州金琪基因技术研究发展中心 | Method for screening external-direction sequence libraries |
| CN102178690A (en) * | 2011-01-14 | 2011-09-14 | 泰州市病毒研究所 | Preparation method of EGS nucleic acid drug for resisting influenza virus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5683873A (en) * | 1995-01-13 | 1997-11-04 | Innovir Laboratories, Inc. | EGS-mediated inactivation of target RNA |
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2009
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101634047A (en) * | 2009-08-12 | 2010-01-27 | 广州金琪基因技术研究发展中心 | Method for screening external-direction sequence libraries |
| CN102178690A (en) * | 2011-01-14 | 2011-09-14 | 泰州市病毒研究所 | Preparation method of EGS nucleic acid drug for resisting influenza virus |
| CN102178690B (en) * | 2011-01-14 | 2013-10-02 | 泰州市病毒研究所 | Preparation method of EGS nucleic acid drug for resisting influenza virus |
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