CN102533831A - Tissue-specific double gene silencing RNAi expression vector having self-splicing locus - Google Patents

Tissue-specific double gene silencing RNAi expression vector having self-splicing locus Download PDF

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CN102533831A
CN102533831A CN2012100339350A CN201210033935A CN102533831A CN 102533831 A CN102533831 A CN 102533831A CN 2012100339350 A CN2012100339350 A CN 2012100339350A CN 201210033935 A CN201210033935 A CN 201210033935A CN 102533831 A CN102533831 A CN 102533831A
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restriction enzyme
sequence
carrier
shrna
site
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CN102533831B (en
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任雪玲
张振中
柴丽娜
宋雨
张云
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Zhengzhou University
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Zhengzhou University
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Abstract

The invention relates to a tissue-specific double gene silencing RNAi expression vector having a self-splicing locus and solves the problem of limitation on the RNAi expression vector inhibiting two genes at the same time during transfection of a mammalian cell and the problem of non-tissue-specific action in a gene silencing process. The vector has a tissue-specific double gene silencing shRNA expression cassette. The tissue-specific double gene silencing shRNA expression cassette comprises multiple cloning sites to be inserted into a tissue-specific promoter, two ribozyme sequences containing self-splicing loci, two multiple cloning sites to be inserted into an shRNA template sequence and a transcription terminator sequence.

Description

A kind of comprising from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site
Technical field
The present invention relates to medical field, particularly a kind of comprising from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site.
Background technology
The RNA interference (RNA interference; RNAi) be meant double-stranded (the short interfering RNA of the little RNA with particular sequence; SiRNA) behind the transporte to cells through inducing homology messenger RNA(mRNA) (messenger RNA; MRNA) degrade, cause the phenomenon (Fire et al, Nature 1998391:806) of specific gene generation PTGS.SiRNA has the Yeast Nucleic Acid of 3 ' redundant C-terminal of two bases (this molecular energy combines with reticent mixture and target gene mRNA is cut for ribonucleic acid, RNA) duplex molecule.Cutting usually occurs in the centre of siRNA homology part, from 10 base places of siRNA antisense strand 5 ' end.SiRNA not only is prone to by the RNA enzyme liberating that extensively exists in the environment, and have poor stability, restraining effect of short duration, can not heredity, shortcoming (Emily et al, Methods in Molecular Biology 2008442:45) such as cost height.Therefore; The investigator has been developed by RNA polymerase (RNA polymerase; Pol) transcribe the method that obtains siRNA in vivo; Usually (short hairpin RNA shRNA) produces (Paddison et al, Genes & Development 2002 16:948) to this method by short hairpin RNA.ShRNA is folded through self by a RNA single strand to form, and comprises the inverted repeats of two weak points, and the centre is separated by stem ring (loop) sequence, forms similar hairpin structure.ShRNA is transcribed by expression vector and obtains, and can imitate the duplex structure of siRNA, can be in vivo, the expression of external effective inhibition target gene mRNA, realize the RNAi effect.Because therefore series of advantages such as shRNA has can be hereditary, with low cost, and structure is more stable have become the important tool of studying gene function, and have played a significant role in fields such as therapy of tumor, inherited disease researchs.
At present, the shRNA expression vector is normally by rna plymerase iii (Pol III) promoter regulation, for example H1, U6 promotor.Pol III promotor has clear and definite transcription initiation site and Transcription Termination site (TTTTTT), can realize efficient and stable gene silencing in vivo.But the inorganizable specificity of Pol III promotor, the also inorganizable specificity of RNAi effect that its regulation and control produce.Tissue specificity Pol II promotor meeting regulatory transcription product is in particular organization's cell and intraorganic expression.For example; PSA (PSA) promotor can make transcription product only in prostatic cell, express (Zhao et al; Seminars and Original Investigations 200927:539); Apolipoprotein A-1 (apolipoprotein A-I, apoA-I) promotor has hepatic tissue specificity (Ren Xueling etc., contemporary Chinese medical journal 200922:3403); HTRT (Human telomerase reverse transcnptase; HTERT) promotor can be transcribed by regulatory gene in tumour cell, and is reticent (Takeda et al, The Journal of Clinical Endocrinology & Metabolism 200888:3531) or the like in non-tumor cell.Therefore, utilize Pol II promotor to realize that the tissue-specific transcription of shRNA is the available strategy that solves RNAi effect tissue specificity problem.
Pol II promotor can realize the tissue specificity of transcription product, but its transcriptional regulatory mechanism is obviously different with Pol III promotor.In a single day at first, Pol II promotor does not have clear and definite transcription initiation site, and transcribes beginning, newly-generated mRNA can add inverse guanosine nucleotide triphosphoric acid at 5 ' end, and produces m through further methylation 7The G cap sequence.Secondly, Pol II promotor does not have clear and definite Transcription Termination site yet, and its Transcription Termination occurs in after several kilobase in 3 ' end downstream of ripe transcription product.Transcription product adds the VITAMIN B4 that quantity does not wait through inscribe and further polyadenylation at 3 ' end, forms the tail of poly VITAMIN B4 (polyA).In the Pol II promoter regulation mechanism 5 ' end m 7G cap sequence and 3 ' end poly A tail are extremely important for the function of transcription product, are not only one of principal element that influences transcription product stability, also promote transcription product to get into endochylema through nucleopore rapidly simultaneously.When the transcription product of Pol II promoter regulation generation was shRNA, shRNA rapidly, constantly got into the uncertainty that will cause the RNAi effect in the endochylema, the effect of for example missing the target.Therefore, effectively remove structural 5 ' the end m of shRNA that Pol II promoter regulation produces 7G cap and 3 ' end poly A tail are most important for inducing effective and stable RNAi effect.
Ribozyme (Ribozyme) is one type of RNA molecule with catalysis, can participate in RNA self shearing, the course of processing through changeing SULPHOSUCCINIC ACID ESTER and phosphodiester bond hydrolysis reaction.In these have the ribozyme of catalytic activity, have some molecules can be from carrying out autocatalysis as substrate, with the excision of the redundance in the RNA molecule, we claim that this quasi-molecule is for from shearing ribozyme.Investigators such as Shinagawa will shear ribozyme certainly and be inserted between cytomegalovirus promoter and the shRNA sequence; Excise the cap sequence (Shinagawa et al, Genes & Development 200317:1340) of transcription product 5 ' end through the self-shearing action of ribozyme.Zinc finger protein MAZ binding sequence can effectively stop transcribing, owing to lack tailing signal in the sequence, and therefore can be at 3 ' the terminal polyA tail (Yonaha et al, The EMBO Journal 200019:3770) that forms of transcription product.Therefore, as the shRNA terminator sequence, can effectively remove the polyA tail in the shRNA structure, get into cytoplasmic speed by nucleus, increase the stability and the efficient of RNAi effect thereby slowed down transcription product with zinc finger protein MAZ binding sequence.
In body, all vital processes all are a kind of processes of complicacy, are the results of multifactor, polygene, the comprehensive action of many conditions.Based on cellular and molecular level research, intracellular each bar path all relates to a plurality of genes, and each gene possibly exist in a plurality of different paths.For example, in the molecular mechanism of drug habit, relate to a plurality of molecules (Angel et al, Biochim Biophys Acta 19911072:129) such as proto-oncogene c-fos, proto-oncogene JunB; Human epidermal growth factor receptor (Human epidermal growth factor receptor; HEGFR) at ultraviolet radiation damage (Ann et al; Science Signaling), has keying action in tumor development a plurality of mechanism such as (Ann et al, Nature Review Cancer 20099:508).Therefore, target is difficult to reach dreamboat in the RNAi of individual gene technology at aspects such as gene functional research, gene therapies.With the tumour is example; The most of tumour of research proof is not by single path domination; Its each stage needs two or more different gene unconventionality inactivations relevant with cancer at least or activates, and different gene then is in a molecular network system that is interweaved, works in coordination with each other.So only from an aspect or a gene start with, often do not reach the expection cancer resistant effect, thoroughly anticancer growth, tumour also just can't obtain effecting a radical cure.The RNAi technology can effectively suppress the expression of goal gene, has been widely used in the gene therapy of the multiple disease that comprises tumour.At present existing investigator is according to key gene design shRNA different in the tumor development process, and the RNAi expression vector that makes up target different carcinoma genes involved is used for the experimental research of therapy of tumor.Also there is the investigator to recognize the importance and the necessity of a plurality of gene Synergistic treatments, attempts adopting associating RNAi technology to be used for therapy of tumor (Daniel et al, TRENDS in Biotechnology 2006 24:76).But the investigator makes up the RNAi expression vector of target individual gene usually respectively, adopts the method for cotransfection then, and with the different common transfection tumor cells of RNAi expression vector, and then research associating gene silencing is for the effect of therapy of tumor.Because the RNAi expression vector has strict requirement to its consumption when transfection mammalian cell, this has just limited the RNAi effect to a certain extent.Therefore,, some experiment often receives very big restriction when especially need suppressing two genes simultaneously.
Summary of the invention
To above-mentioned situation; For overcoming the prior art defective; The present invention's purpose is exactly will provide a kind of to comprise from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site, can effectively solve be restricted when the RNAi expression vector suppresses two genes simultaneously when transfection mammalian cell and the gene silencing process in the problem of non-tissue specificity effect is arranged.
The technical scheme that the present invention solves is, this carrier contain a dual-gene reticent shRNA expression cassette of tissue specificity (as shown in Figure 1, ts-P wherein Pol IIBe meant to have tissue-specific rna plymerase ii promotor, Ribo-R is meant from shearing ribozyme sequence, promptly contains the ribozyme sequence from shearing site; MCS is meant MCS, and TSS is meant the sequence with Transcription Termination function, and shRNA is meant the shRNA template sequence; I wherein, II, III; IV, V, VI; VII refers to different restriction enzymes respectively, representes the restriction enzyme site of this enzyme with arrow, the MCS that wherein inserts as tissue-specific promoter with the restriction enzyme site of I and II; Represent that with MCS-1 the restriction enzyme site of restriction enzyme III and IV forms the MCS that first shRNA template sequence inserts, and representes with MCS-2; First shRNA template sequence representes that with shRNA-1 the restriction enzyme site of restriction enzyme V and VI forms the MCS that second shRNA template sequence inserts, and representes with MCS-3; Second shRNA template sequence represented with shRNA-2), the dual-gene reticent shRNA expression cassette of described tissue specificity comprises: MCS that inserts tissue-specific promoter, two contain the ribozyme sequence from shearing site, two MCS and transcription termination sequences that insert the shRNA template sequences; The MCS of described insertion tissue-specific promoter is arranged in two and contains from first of the ribozyme sequence of shearing site and contain the ribozyme sequence upper reaches from shearing site; Described transcription termination sequence is arranged in two second ribozyme sequence downstream of containing from shearing site containing from the ribozyme sequence of shearing site; First MCS that inserts the shRNA template sequence in the MCS of described two insertion shRNA template sequences contains between the ribozyme sequence of shearing site at two, and second MCS that inserts the shRNA template sequence contains between the ribozyme sequence and transcription termination sequence of shearing site at second; Described MCS is meant the section of DNA sequence that comprises two or more restriction enzyme digestion sites;
A kind of construction process that comprises from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site, realize by following steps:
(1) adds respectively and the restriction enzyme II of initial carrier and the identical restriction enzyme site of restriction enzyme site of III from the 5 ' end of shearing ribozyme sequence and 3 ' end at first; Synthetic; Winning has the oligonucleotide from shearing ribozyme sequence, with restriction enzyme II and III double digestion, is inserted into the identical restriction enzyme site of initial carrier; Adopt ordinary method to make up and contain first from the carrier A of shearing ribozyme sequence; Described first contains one from shearing site from shearing on ribozyme sequence, correspondingly on the carrier A also contain first from shearing site, and initial carrier is for inserting nucleic acid fragment, can carrying exogenous nucleic acid and get into mammalian cell; And the nucleic acid molecule that carries out independent and stable self-replacation therein, like initial carrier pEGFP-C1;
(2) at second from 5 ' terminal and 3 ' terminal add respectively and first of carrier A sheared the restriction enzyme IV in ribozyme sequence downstream and the identical restriction enzyme site of restriction enzyme site of V certainly of shearing ribozyme sequence; Synthetic; Getting second has from the oligonucleotide of shearing ribozyme sequence; With restriction enzyme IV and V double digestion; Be inserted into the identical restriction enzyme site of carrier A; Adopt ordinary method to make up to contain first from shear ribozyme sequence and second from shear ribozyme sequence totally two described second contains one from shearing site from shearing on the ribozyme sequence from the carrier B of shearing ribozyme sequence, so on the carrier B corresponding contain first from shearing site and second from shearing site totally two from shearing site;
(3) shear the restriction enzyme VI in ribozyme sequence downstream and the identical restriction enzyme site of restriction enzyme site of VII certainly at 5 ' end of transcription termination sequence and 3 ' terminal interpolation respectively and carrier B second; Synthetic; The oligonucleotide that must have transcription termination sequence; With restriction enzyme VI and VII double digestion; Be inserted into the identical restriction enzyme site of carrier B; Adopt ordinary method to make up to contain first from shear ribozyme sequence and second from shear ribozyme sequence totally two from the support C of shearing a ribozyme sequence and a transcription termination sequence; Be and contain the comprising from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site of a dual-gene reticent shRNA expression cassette of tissue specificity, the restriction enzyme II restriction enzyme site upper reaches of said support C have restriction enzyme I restriction enzyme site, and restriction enzyme I and II restriction enzyme site are as the MCS that inserts tissue-specific promoter; Restriction enzyme III and IV restriction enzyme site insert the MCS of shRNA template sequence as first, and restriction enzyme V and VI restriction enzyme site are as second MCS that inserts the shRNA template sequence; The oligonucleotide that has from the oligonucleotide of shearing ribozyme sequence and synthetic transcription termination sequence of described synthetic provides completion by Beijing three rich polygala root biotech companies.
Annotate: " structure " described in this specification sheets carrier, belong to prior art, ([U.S.A] J. Sa nurse Brooker and D.W. Russell, Huang Peitang etc. translate, molecular cloning experiment guide (third edition) with reference to " molecular cloning experiment guide " in concrete operations.Beijing: Science Press, 2002).
Contain application from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site:
(1) at 5 ' terminal restriction enzyme III identical with first shRNA template sequence MCS of support C of first shRNA template sequence and the restriction enzyme site of IV with 3 ' terminal interpolation respectively; Synthetic; Behind restriction enzyme III and IV double digestion; Be inserted into the MCS place of containing first insertion shRNA template sequence in the dual-gene reticent shRNA expression cassette of tissue specificity in the support C; Adopt ordinary method carrier construction D; At 5 ' terminal restriction enzyme V identical with second shRNA template sequence MCS of carrier D with 3 ' terminal interpolation respectively of second shRNA template sequence and the restriction enzyme site of VI, synthetic is behind restriction enzyme V and VI double digestion; Be inserted into second MCS place that inserts the shRNA template sequence among the carrier D, adopt ordinary method carrier construction E;
Synthetic be used to the to increase primer of tissue-specific promoter; Genomic dna with human archeocyte is that template is carried out pcr amplification; The PCR product that must have tissue-specific promoter's sequence; The PCR product that will have tissue-specific promoter's sequence with and the restriction enzyme I of tissue-specific promoter's MCS of carrier E and corresponding restriction enzyme I of restriction enzyme site and the II double digestion of II; Be inserted into tissue-specific promoter's MCS of carrier E, adopt ordinary method to make up two heterogeneic carrier F that contain the dual-gene reticent shRNA expression cassette of tissue specificity of target; Or the MCS place that in support C, inserts tissue-specific promoter inserts earlier through pcr amplification; Tissue-specific promoter behind the double digestion; Adopt ordinary method carrier construction G; The MCS place that in carrier G first inserts the shRNA template sequence inserts synthetic again after first shRNA template sequence of double digestion; Adopt ordinary method carrier construction H, second MCS place that inserts the shRNA template sequence in carrier H inserts synthetic after second shRNA template sequence of double digestion adopts ordinary method to make up two heterogeneic carrier R that contain the dual-gene reticent shRNA expression cassette of tissue specificity of target; Described human archeocyte is people's such as human liver cancer cell SMMC-7721 a cell;
(2) or with the carrier F that contains the dual-gene reticent shRNA expression cassette of tissue specificity or the dual-gene reticent shRNA expression cassette of the tissue specificity among the carrier R of above-mentioned structure be inserted among other different carrier K, can adopt ordinary method to make up different containing from the dual-gene reticent RNAi expression vector H of the tissue specificity of shearing site; Described carrier K is for inserting the exogenous nucleic acid fragment, can carrying exogenous nucleic acid and get into mammalian cell and the nucleic acid molecule that carries out independent and stable self-replacation therein;
Described tissue-specific promoter has tissue-specific rna plymerase ii promotor, comprises and has the specific hTRT's promotor of tumor tissues (claim not only hTERT promotor) SEQ ID NO:25 or have the specific apolipoprotein A-1 promotor of hepatic tissue (but also claiming the apoA-I promotor) SEQ ID NO:24; Described is the site (as shown in Figure 2) that self-shearing action takes place from shearing in the ribozyme sequence from shearing site, shears ribozyme sequence certainly and comprises cis acting Hammerhead ribozyme sequence (being called for short the ribozyme sequence) SEQ ID NO:1; Described transcription termination sequence is to stop the sequence that DNA transcribes, and comprises zinc finger protein MAZ binding sequence (being called for short the MAZ sequence) SEQ ID NO:2; Described two shRNA template sequences are meant through transcribing and can obtain shRNA; And induce target gene that the sequence of RNAi effect takes place, comprise any two among shRNA template sequence (being called for short JunB shRNA sequence) the SEQ ID NO:4 of shRNA template sequence (being called for short c-fos shRNA sequence) SEQ ID NO:6, target proto-oncogene JunB of shRNA template sequence (being called for short hEGFR shRNA sequence) SEQ ID NO:5, the targeted cells proto-oncogene c-fos of target hTRT's shRNA template sequence (being called for short hTERT shRNA sequence) SEQ ID NO:3, target Human epidermal growth factor receptor.
Construction process of the present invention is simple, and is easy to operate, can replace the heterogeneic shRNA template sequence of different tissue-specific promoters or target as required, can construct to have from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site.
Description of drawings
Fig. 1 is a shRNA expression cassette structural representation on the dual-gene reticent RNAi expression vector of tissue specificity.
Fig. 2 is cis acting Hammerhead ribozyme template sequence and site that self-shearing action takes place.
The dual-gene reticent RNAi expression vector pTSDS structural representation of Fig. 3 tissue specificity.
Embodiment
Elaborate below in conjunction with the embodiment specific embodiments of the invention.
1, bacterial strain and carrier: initial carrier pEGFP-C1 (the GenBank accession number is U55763) is available from BD Biosciences Clontech company; Carrier pRS is available from OriGene company, and sequence is shown in http://www.origene.com/rna/vector_information.mspx; Liver cancer cell SMMC-7721, lung cell A549, esophageal cancer cell EC9706, human embryonic lung cell WI-38 (the 12nd generation) is all available from Chinese Academy of Sciences's cell bank.
2, enzyme and reagent: restriction enzyme (Ase I, Nhe I, Bgl II, HindIII, EcoR I, Kpn I, BamH I), T 4Dna ligase, T 4Enzyme commonly used such as archaeal dna polymerase is available from Fermentas company; Sepharose DNA reclaims test kit available from TIANGEN Biotech (Beijing) Co., Ltd.; High sugared DMEM substratum, foetal calf serum are available from U.S. HyClone company; In-vitro transfection reagent E xGen 500 is available from Fermentas company; Lipofectamine Lipofectamine TM2000 available from Invitrogen company, and all primers, the synthetic gene sequencing that reaches of oligonucleotide provide completion by Beijing three rich polygala root biotech companies.
3, the DMEM substratum of 10% foetal calf serum: in the 900mlDMEM substratum, add the 100ml foetal calf serum and process.
4, the nucleotide sequence of using in the test, used sequence is not used for limiting scope of the present invention.
(1) be used for the primer of reverse transcriptase polymerase chain reaction (RT-PCR):
Specificity hTERT primer: upstream primer, 5 '-cggaagagtgtctggagcaa-3 '; Downstream primer, 5 '-ggatgaagcggagtctgga-3 '.Specific beta-actin primer: upstream primer, 5 '-ctgggacgacaggagaaaa-3 '; Downstream primer, 5 '-aaggaaggctggaagagtgc-3 '.Specificity hEGFR primer: upstream primer, 5 '-agctcacgcagttgggcact-3 '; Downstream primer, 5 '-tctcatgggcagctccttca-3 '.Specificity c-fos primer: upstream primer, 5 '-ccgactccttctccagcat-3 '; Downstream primer, 5 '-tcaccgtggggataaagttg-3 '.Specificity JunB primer: upstream primer, 5 '-atgtgcactaaaatggaaca-3 '; Downstream primer, 5 '-cctgaccagaaaagtagctg-3 '.
(2) be used for the primer of polymerase chain reaction (PCR) amplification promotor:
The primer of apolipoprotein A-1 promotor is used to increase: upstream primer, 5 '-ATTAATaggggaaggggatgagtg-3 '; Downstream primer, 5 '-GCTAGCgagaagaagggcctggctga-3 '.
The primer of hTERT promotor is used to increase: upstream primer, 5 '-ATTAATtccgcccggagcagctgcg-3 '; Downstream primer, 5 '-GCTAGCgcagcactcgggccaccag-3 '.
Explain: carrier construction for ease, on the promotor amplimer, increase restriction enzyme site, represent that with capitalization what wherein add on the upstream primer is Ase I restriction enzyme site, what on downstream primer, add is Nhe I restriction enzyme site.
Embodiment 1:
(1) first has from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert initial carrier pEGFP-C1 for ease; 5 ' the terminal Nhe I restriction enzyme site (GCTAGC) that adds at first cis acting Hammerhead ribozyme sequence (SEQ ID NO:1); At 3 ' the terminal Bgl II restriction enzyme site (AGATCT) that adds; Synthetic, an oligonucleotide with cis acting Hammerhead ribozyme sequence of winning is double-stranded, with restriction enzyme Nhe I and Bgl II double digestion; Insert Nhe I and the Bgl II restriction enzyme site of initial carrier pEGFP-C1, adopt ordinary method carrier construction pRib (SEQ ID NO:7) thus;
(2) second have from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert carrier pRib for ease; 5 ' the terminal HindIII restriction enzyme site (AAGCTT) that adds at second cis acting Hammerhead ribozyme sequence (SEQ ID NO:1); 3 ' the terminal EcoR I restriction enzyme site (GAATTC) that adds; Synthetic, it is double-stranded to get second oligonucleotide with cis acting Hammerhead ribozyme sequence, with restriction enzyme HindIII and EcoR I double digestion; First that is inserted into carrier pRib adopts ordinary method carrier construction pDi-Rib (SEQ ID NO:8) thus from shearing the HindIII and the EcoR I restriction enzyme site in ribozyme sequence downstream;
(3) have the insertion of the oligonucleotide of transcription termination sequence:
Insert carrier pDi-Rib for ease; 5 ' the terminal Kpn I restriction enzyme site (GGTACC) that adds at zinc finger protein MAZ binding sequence (SEQ ID NO:2); 3 ' the terminal BamH I restriction enzyme site (GGATCC) that adds; Synthetic, the oligonucleotide that must have zinc finger protein MAZ binding sequence is double-stranded, with restriction enzyme Kpn I and BamH I double digestion; Certainly shear the Kpn I and the BamH I restriction enzyme site in ribozyme sequence downstream, adopt ordinary method carrier construction pTSDS (SEQ ID NO:9) thus for second that is inserted into carrier pDi-Rib; As shown in Figure 3, in Fig. 3, listed the structural representation of carrier pTSDS, wherein ts-P Pol IIBe meant tissue specificity rna plymerase ii promotor; Ribo-c is meant cis acting Hammerhead ribozyme sequence; MAZ is meant zinc finger protein MAZ binding sequence; ShRNA-1 is meant first shRNA template sequence; ShRNA-2 is meant second shRNA template sequence, and wherein Ase I, Nhe I, Bgl II, HindIII, EcoR I, Kpn I are meant the restriction enzyme site of restriction enzyme A se I, Nhe I, Bgl II, HindIII, EcoR I, Kpn I, has Ase I restriction enzyme site at the Nhe of the carrier pTSDS I restriction enzyme site upper reaches; Formed the MCS that tissue-specific promoter inserts between Ase I and the Nhe I restriction enzyme site; Can have tissue-specific rna plymerase ii promotor in this site insertion, form the MCS that first shRNA template sequence inserts between Bgl II and the HindIII restriction enzyme site, can insert the shRNA template sequence of first target gene in this site; Formed second MCS that the shRNA template sequence inserts between EcoR I and the Kpn I restriction enzyme site, can insert the shRNA template sequence of second target gene in this site;
(4) first shRNA template sequence MCS inserts hTERT shRNA sequence:
Insert carrier pTSDS for ease; 5 ' the terminal Bgl II restriction enzyme site that adds in hTERT shRNA sequence (SEQ ID NO:3); 3 ' the terminal HindIII restriction enzyme site that adds, synthetic, the oligonucleotide that must contain hTERT shRNA sequence is double-stranded; With restriction enzyme Bgl II and HindIII double digestion; Be inserted into the MCS of first shRNA template sequence of carrier pTSDS after enzyme is cut, promptly Bgl II and HindIII restriction enzyme site adopt ordinary method to make up the carrier pTSDS-t (SEQ ID NO:10) that obtains target hTERT thus;
(5) second shRNA template sequence MCSs insert JunB shRNA sequence:
Insert carrier pTSDS-t for ease; 5 ' the terminal EcoR I restriction enzyme site that adds in JunB shRNA sequence (SEQ ID NO:4); 3 ' the terminal Kpn I restriction enzyme site that adds, synthetic, the oligonucleotide that must contain JunB shRNA sequence is double-stranded; With restriction enzyme EcoR I and Kpn I double digestion; Be inserted into the MCS of second shRNA template sequence of carrier pTSDS-t after enzyme is cut, promptly EcoR I and Kpn I restriction enzyme site adopt ordinary method to make up the carrier pTSDS-tb (SEQ ID NO:11) that obtains target hTERT and JunB thus;
(6) amplification of apoA-I promotor, and apoA-I promotor is inserted tissue-specific promoter's MCS:
Synthetic be used to the to increase primer of apoA-I promotor: upstream primer 5 '-ATTAATaggggaaggggatgagtg-3 '; Downstream primer 5 '-GCTAGCgagaagaagggcctggctga-3 '; With human liver cancer cell SMMC-7721 genome is that template is carried out pcr amplification; Obtain having the apoA-I promoter sequence PCR product of (SEQ ID NO:24); The PCR product that will have the apoA-I promoter sequence is inserted into tissue-specific promoter's MCS of carrier pTSDS-tb with restriction enzyme A se I and Nhe I double digestion, i.e. Ase I and Nhe I restriction enzyme site; Through sequence verification, adopt ordinary method to make up and obtain target hTERT and the dual-gene specific dual-gene reticent RNAi expression vector pTSDS/apo-tb of hepatic tissue (SEQ ID NO:12) of JunB simultaneously;
(7) carrier pTSDS/apo-tb transfection liver cancer cell SMMC-7721 and RNAi effect checking:
Human liver cancer cell SMMC-7721 is at the high sugared DMEM substratum that contains 10% foetal calf serum and 37 ℃, 5%CO 2Carry out routine under the condition and cultivate, 16h is by 1 * 10 before the transfection 6Individual/hole is inoculated in 6 orifice plates, and overnight cultures when cell attachment grows into 90% fusion, is used lipofectamine Lipofectamine TM2000 with the dual-gene silent carrier pTSDS/apo-tb of liver specificity transfection to liver cancer cell SMMC-7721, concrete steps are following: with 40 μ l carrier pTSDS/apo-tb and 10 μ l lipofectamine Lipofectamine TM2000 add in the 500 μ l serum-free DMEM substratum, the vortex mixing, and room temperature leaves standstill 20min; Get mixture; Then this mixture is added human liver cancer cell SMMC-7721, cultivated 6 hours for 37 ℃, add the DMEM substratum that contains 10% foetal calf serum then and continue to cultivate; Controlled trial is set simultaneously, and controlled trial is to adopt identical way with carrier pTSDS/apo-tb transfection human esophagus cancer cell EC9706;
Behind the transfection 72h; Tetramethyl-azo azoles salt colourimetry (mtt assay) is measured cell proliferation; Experimental result shows that the liver cancer cell growth inhibiting rate of transfection carrier pTSDS/apo-tb is 57%, and the growth of the esophageal cancer cell of transfection carrier pTSDS/apo-tb does not receive remarkable inhibition; Adopt the Trizol method to extract cell total rna; Adopt the reticent efficient of RT-PCR technology for detection target gene: with specificity hTERT primer amplification hTERT fragment (SEQ ID NO:19), with specificity JunB primer amplification JunB fragment (SEQ ID NO:22), simultaneously with the expression level of specific beta-actin primer amplification tumour cell house-keeping gene β-actin fragment (SEQ ID NO:23) as internal control gene; Experimental result shows; The relative expression quantity of hTERT and JunB is respectively 0.24 and 0.31 in the liver cancer cell of transfection carrier pTSDS/apo-tb, i.e. the expression of hTERT and JunB all receives remarkable inhibition, and inhibiting rate is respectively 76% and 69%; The relative expression quantity of hTERT and JunB is respectively 0.94 and 0.99 in the esophageal cancer cell of transfection carrier pTSDS/apo-tb; Considerable change does not all take place in the expression that is hTERT mRNA and hEGFR mRNA, and test-results shows that the dual-gene silent carrier pTSDS/apo-tb of liver specificity can significantly suppress the expression of hTERT and JunB in liver cancer cell, induce the RNAi effect; Suppress cell proliferation, and in non-hepatocellular esophageal cancer cell, can not obviously induce the RNAi effect.
Embodiment 2:
(1) first has from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert initial carrier pEGFP-C1 for ease; 5 ' the terminal Nhe I restriction enzyme site (GCTAGC) that adds at first cis acting Hammerhead ribozyme sequence (SEQ ID NO:1); At 3 ' the terminal Bgl II restriction enzyme site (AGATCT) that adds; Synthetic, an oligonucleotide with cis acting Hammerhead ribozyme sequence of winning is double-stranded, with restriction enzyme Nhe I and Bgl II double digestion; Insert Nhe I and the Bgl II restriction enzyme site of initial carrier pEGFP-C1, adopt ordinary method carrier construction pRib (SEQ ID NO:7) thus;
(2) second have from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert carrier pRib for ease; 5 ' the terminal HindIII restriction enzyme site (AAGCTT) that adds at second cis acting Hammerhead ribozyme sequence (SEQ ID NO:1); 3 ' the terminal EcoR I restriction enzyme site (GAATTC) that adds; Synthetic, it is double-stranded to get second oligonucleotide with cis acting Hammerhead ribozyme sequence, with restriction enzyme HindIII and EcoR I double digestion; First that is inserted into carrier pRib adopts ordinary method carrier construction pDi-Rib (SEQ ID NO:8) thus from shearing the HindIII and the EcoR I restriction enzyme site in ribozyme sequence downstream;
(3) have the insertion of the oligonucleotide of transcription termination sequence:
Insert carrier pDi-Rib for ease; 5 ' the terminal Kpn I restriction enzyme site (GGTACC) that adds at zinc finger protein MAZ binding sequence (SEQ ID NO:2); 3 ' the terminal BamH I restriction enzyme site (GGATCC) that adds; Synthetic, the oligonucleotide that must have zinc finger protein MAZ binding sequence is double-stranded, with restriction enzyme Kpn I and BamH I double digestion; Certainly shear the Kpn I and the BamH I restriction enzyme site in ribozyme sequence downstream, adopt ordinary method carrier construction pTSDS (SEQ ID NO:9) thus for second that is inserted into carrier pDi-Rib;
(4) amplification of hTERT promotor, and hTERT promotor is inserted tissue-specific promoter's MCS:
Synthetic be used to the to increase primer of hTERT promotor: upstream primer 5 '-ATTAATtccgcccggagcagctgcg-3 '; Downstream primer; 5 '-GCTAGCgcagcactcgggccaccag-3 '; With human liver cancer cell SMMC-7721 genome is that template is carried out pcr amplification, and obtain having the hTERT promoter sequence PCR product of (SEQ ID NO:25) is with restriction enzyme A se I and Nhe
The I double digestion; After cutting, enzyme is inserted into tissue-specific promoter's MCS of carrier pTSDS; Be Ase I and Nhe I restriction enzyme site,, adopt ordinary method to make up the dual-gene reticent RNAi expression vector pTSDS/tert (SEQ ID NO:13) that obtains having tumour-specific through sequence verification;
(5) first shRNA template sequence MCS inserts hEGFR shRNA sequence:
Insert carrier pTSDS/tert for ease; 5 ' the terminal Bgl II restriction enzyme site that adds in hEGFR shRNA sequence (SEQ ID NO:5); 3 ' the terminal HindIII restriction enzyme site that adds, synthetic, the oligonucleotide that must contain hEGFR shRNA sequence is double-stranded; With restriction enzyme Bgl II and HindIII double digestion; Be inserted into the MCS of first insertion shRNA template sequence of carrier pTSDS/tert after enzyme is cut, promptly Bgl II and HindIII restriction enzyme site adopt ordinary method to make up the carrier pTSDS/tert-e (SEQ ID NO:14) that obtains target hEGFR thus;
(6) second shRNA template sequence MCSs insert c-fos shRNA sequence:
Insert carrier pTSDS/tert-e for ease; 5 ' the terminal EcoR I restriction enzyme site that adds in c-fos shRNA sequence (SEQ ID NO:6); 3 ' the terminal Kpn I restriction enzyme site that adds; Synthetic, the oligonucleotide that must contain c-fos shRNA sequence is double-stranded, with restriction enzyme EcoR I and Kpn I double digestion; Be inserted into EcoR I and pn I restriction enzyme site in the MCS of second shRNA template sequence of carrier pTSDS/tert-e after enzyme is cut, adopt ordinary method to make up to obtain the dual-gene silent carrier pTSDS/tert-ef of tumor tissues specificity (SEQ ID NO:15) of target c-fos and hEGFR simultaneously;
(7) structure of the dual-gene silencing virus carrier of tumor tissues specificity:
With the dual-gene silent carrier pTSDS/tert-ef of the tumour-specific of while target c-fos and hEGFR with restriction enzyme A se I and BamH I double digestion, and with T 4Archaeal dna polymerase is mended sticky end flat, and agarose gel electrophoresis reclaims small segment (SEQ ID NO:17), with restriction enzyme EcoR I and HindIII double digestion retroviral vector pRS (OriGene), and with T 4Archaeal dna polymerase is mended sticky end flat, and agarose gel electrophoresis reclaims big fragment (SEQ ID NO:18), with T 4Dna ligase is connected above-mentioned small segment with big fragment, adopt ordinary method to make up and obtain the dual-gene silencing virus carrier of tumor tissues specificity pTSDS/MMLV/tert-ef (SEQ ID NO:16);
(8) carrier pTSDS/MMLV/tert-ef cell transfecting and RNAi effect checking:
Lung cell A549 is at the DMEM substratum that contains 10% foetal calf serum and 37 ℃, 5%CO 2Carry out routine under the condition and cultivate, 16h is by 7.25 * 10 before the transfection 5Individual/hole is inoculated in 6 orifice plates; Overnight cultures; Cell attachment grows into 60% when merging, with in-vitro transfection reagent E xGen 500 with the dual-gene silent carrier pTSDS/MMLV/tert-ef of tumour-specific transfection to lung carcinoma cell, concrete steps are following: 3 μ g carrier pTSDS/MMLV/tert-ef are dissolved among the NaCl of 200 μ l 150mmol/L; The vortex mixing; Add the rapid vortex 10s of 9.87 μ l transfection reagent ExGen500, room temperature incubation 10min, the mixture with transfection reagent ExGen500 and carrier pTSDS/MMLV/tert-ef adds in the substratum then; Controlled trial is set simultaneously, and controlled trial is the human embryonic lung cell WI-38 (the 12nd generation) with carrier pTSDS/MMLV/tert-ef transfection non-tumor cell system;
Simultaneously behind the dual-gene dual-gene silencing virus carrier of the tumour-specific pTSDS/MMLV/tert-ef transfection 48h of target hEGFR and c-fos; Mtt assay is measured cell proliferation; Experimental result shows; The lung carcinoma cell growth inhibition ratio of transfection carrier pTSDS/MMLV/tert-ef is 57%; And the human embryonic lung cell of transfection carrier pTSDS/MMLV/tert-ef growth does not receive remarkable inhibition, adopts the Trizol method to extract cell total rna, adopts the reticent efficient of RT-PCR technology for detection target gene: with specificity hEGFR primer amplification hEGFR fragment (SEQ ID NO:20); With specificity c-fos primer amplification c-fos fragment (SEQ ID NO:21); With the expression level of specific beta-actm primer amplification β-actm fragment (SEQ ID NO:23) as internal control gene, experimental result shows that the relative expression quantity of c-fos and hEGFR is respectively 0.31 and 0.19 in the lung carcinoma cell of transfection carrier pTSDS/MMLV/tert-ef simultaneously; The expression that is c-fos and hEGFR all receives remarkable inhibition; Inhibiting rate is respectively 69% and 81%, and the relative expression quantity of hEGFR and c-fos is respectively 0.94 and 0.97 among the human embryonic lung cell of transfection carrier pTSDS/MMLV/tert-ef, i.e. considerable change does not all take place in the expression of c-fos and hEGFR; Test-results shows that the dual-gene silent carrier pTSDS/MMLV/tert-ef of tumour-specific can significantly suppress the expression of c-fos and hEGFR in lung carcinoma cell; Induce the RNAi effect, suppress cell proliferation, and in the human embryonic lung cell of non-tumor cell system, can not obviously induce the RNAi effect.
Show that by above-mentioned carrier of the present invention is tested through repeated multiple times, all obtained satisfied useful technique effect; Fully prove; The present invention is the tissue specificity RNAi expression vector that can be used for dual-gene silence, has overcome the known defective of shRNA carrier, obtains two independently shRNA transcriptons simultaneously through a transcriptional units; Realize the tissue specificity of gene silencing simultaneously, to reduce the nonspecific action in the gene silencing process.The present invention utilizes from shearing ribozyme and makes up two heterogeneic dual-gene RNAi expression vectors of target simultaneously, and uses it for field of gene.Specific strategy is exactly, and between promotor and first shRNA sequence, and inserts one between two shRNA sequences respectively from shearing ribozyme sequence, and the oneself through ribozyme in the transcription product shears function and not only excises 5 ' terminal m 7The G cap separates two shRNA simultaneously.Simultaneously, the present invention adopts to have tissue-specific Pol II promotor in the process that makes up dual-gene RNAi carrier, thus the tissue specificity that regulation and control shRNA expresses, and then realize tissue-specific dual-gene silence, be an innovation greatly of field of medicaments.
Figure IDA0000136147010000011
Figure IDA0000136147010000021
Figure IDA0000136147010000041
Figure IDA0000136147010000051
Figure IDA0000136147010000061
Figure IDA0000136147010000081
Figure IDA0000136147010000091
Figure IDA0000136147010000101
Figure IDA0000136147010000121
Figure IDA0000136147010000131
Figure IDA0000136147010000141
Figure IDA0000136147010000151
Figure IDA0000136147010000161
Figure IDA0000136147010000171
Figure IDA0000136147010000181
Figure IDA0000136147010000201
Figure IDA0000136147010000211
Figure IDA0000136147010000221
Figure IDA0000136147010000231

Claims (6)

1. one kind comprises from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site; It is characterized in that; This carrier contains a dual-gene reticent shRNA expression cassette of tissue specificity, and described this expression cassette comprises: MCS that inserts tissue-specific promoter, two contain the ribozyme sequence from shearing site, two MCS and transcription termination sequences that insert the shRNA template sequences; The MCS of described insertion tissue-specific promoter is arranged in two and contains from first of the ribozyme sequence of shearing site and contain the ribozyme sequence upper reaches from shearing site; Described transcription termination sequence is arranged in two second ribozyme sequence downstream of containing from shearing site containing from the ribozyme sequence of shearing site; First MCS that inserts the shRNA template sequence in the MCS of described two insertion shRNA template sequences contains between the ribozyme sequence of shearing site at two, and second MCS that inserts the shRNA template sequence contains between the ribozyme sequence and transcription termination sequence of shearing site at second;
Comprise structure, realize by following steps from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site:
(1) adds respectively and the restriction enzyme II of initial carrier and the identical restriction enzyme site of restriction enzyme site of III from the 5 ' end of shearing ribozyme sequence and 3 ' end at first; Synthetic; Win and have oligonucleotide from shearing ribozyme sequence; With restriction enzyme II and III double digestion, be inserted into the identical restriction enzyme site of initial carrier, adopt ordinary method to make up and contain first from the carrier A of shearing ribozyme sequence; Described first contains one from shearing site from shearing on ribozyme sequence; Correspondingly on the carrier A also contain first from shearing site, initial carrier is for can inserting the exogenous nucleic acid fragment, can carry exogenous nucleic acid and get into mammalian cell, and the nucleic acid molecule that carries out independent and stable self-replacation therein;
(2) at second from 5 ' terminal and 3 ' terminal add respectively and first of carrier A sheared the restriction enzyme IV in ribozyme sequence downstream and the identical restriction enzyme site of restriction enzyme site of V certainly of shearing ribozyme sequence; Synthetic; Getting second has from the oligonucleotide of shearing ribozyme sequence; With restriction enzyme IV and V double digestion; Be inserted into the identical restriction enzyme site of carrier A; Adopt ordinary method to make up to contain first from shear ribozyme sequence and second from shear ribozyme sequence totally two described second contains one from shearing site from shearing on the ribozyme sequence from the carrier B of shearing ribozyme sequence, so on the carrier B corresponding contain first from shearing site and second from shearing site totally two from shearing site;
(3) shear the restriction enzyme VI in ribozyme sequence downstream and the identical restriction enzyme site of restriction enzyme site of VII certainly at 5 ' end of transcription termination sequence and 3 ' terminal interpolation respectively and carrier B second; Synthetic; The oligonucleotide that must have transcription termination sequence; With restriction enzyme VI and VII double digestion; Be inserted into the identical restriction enzyme site of carrier B; Adopt ordinary method to make up to contain first from shear ribozyme sequence and second from shear ribozyme sequence totally two from the support C of shearing a ribozyme sequence and a transcription termination sequence; Be and contain the comprising from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site of a dual-gene reticent shRNA expression cassette of tissue specificity, the restriction enzyme II restriction enzyme site upper reaches of said support C have restriction enzyme I restriction enzyme site, and restriction enzyme I and II restriction enzyme site are as the MCS that inserts tissue-specific promoter; Restriction enzyme III and IV restriction enzyme site insert the MCS of shRNA template sequence as first, and restriction enzyme V and VI restriction enzyme site are as second MCS that inserts the shRNA template sequence;
Described tissue-specific promoter has tissue-specific rna plymerase ii promotor, comprises having the specific hTRT's promotor of tumor tissues SEQ ID NO:25 or having the specific apolipoprotein A-1 promotor of hepatic tissue SEQID NO:24; Described is the site that self-shearing action takes place from shearing in the ribozyme sequence from shearing site, shears ribozyme sequence certainly and comprises cis acting Hammerhead ribozyme sequence SEQ ID NO:1; Described transcription termination sequence is to stop the sequence that DNA transcribes, and comprises zinc finger protein MAZ binding sequence SEQ ID NO:2; Described two shRNA template sequences are meant through transcribing and can obtain shRNA; And induce target gene that the sequence of RNAi effect takes place, comprise any two among the shRNA template sequence SEQ ID NO:4 of shRNA template sequence SEQ ID NO:6, target proto-oncogene JunB of shRNA template sequence SEQ ID NO:5, the targeted cells proto-oncogene c-fos of target hTRT's shRNA template sequence SEQ ID NO:3, target Human epidermal growth factor receptor.
2. is characterized in that according to claim 1 comprising from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site, realized by following steps:
(1) first has from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert initial carrier pEGFP-C1 for ease; 5 ' the terminal Nhe I restriction enzyme site GCTAGC that adds at first cis acting Hammerhead ribozyme sequence SEQ ID NO:1; At 3 ' the terminal Bgl II restriction enzyme site AGATCT that adds; Synthetic, an oligonucleotide with cis acting Hammerhead ribozyme sequence of winning is double-stranded, with restriction enzyme Nhe I and Bgl II double digestion; Insert Nhe I and the Bgl II restriction enzyme site of initial carrier pEGFP-C1, adopt ordinary method carrier construction pRib SEQ ID NO:7 thus;
(2) second have from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert carrier pRib for ease; 5 ' the terminal HindIII restriction enzyme site AAGCTT that adds at second cis acting Hammerhead ribozyme sequence SEQ ID NO:1; 3 ' the terminal EcoR I restriction enzyme site GAATTC that adds; Synthetic, it is double-stranded to get second oligonucleotide with cis acting Hammerhead ribozyme sequence, with restriction enzyme HindIII and EcoR I double digestion; First that is inserted into carrier pRib adopts ordinary method carrier construction pDi-Rib SEQ ID NO:8 thus from shearing the HindIII and the EcoR I restriction enzyme site in ribozyme sequence downstream;
(3) have the insertion of the oligonucleotide of transcription termination sequence:
Insert carrier pDi-Rib for ease; 5 ' the terminal Kpn I restriction enzyme site GGTACC that adds at zinc finger protein MAZ binding sequence SEQ ID NO:2; 3 ' the terminal BamH I restriction enzyme site GGATCC that adds; Synthetic, the oligonucleotide that must have zinc finger protein MAZ binding sequence is double-stranded, with restriction enzyme Kpn I and BamH I double digestion; Certainly shear the Kpn I and the BamH I restriction enzyme site in ribozyme sequence downstream, adopt ordinary method carrier construction pTSDS SEQ ID NO:9 thus for second that is inserted into carrier pDi-Rib.
3. the described application that comprises from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site of claim 1; Be included in 5 ' terminal restriction enzyme III identical with first shRNA template sequence MCS of support C of first shRNA template sequence and the restriction enzyme site of IV with 3 ' terminal interpolation respectively; Synthetic; Behind restriction enzyme III and IV double digestion; Be inserted into the MCS place of containing first insertion shRNA template sequence in the dual-gene reticent shRNA expression cassette of tissue specificity in the support C, adopt ordinary method carrier construction D, at 5 ' terminal restriction enzyme V identical with second shRNA template sequence MCS of carrier D of second shRNA template sequence and the restriction enzyme site of VI with 3 ' terminal interpolation respectively; Synthetic; Behind restriction enzyme V and VI double digestion, be inserted into second MCS place that inserts the shRNA template sequence among the carrier D, adopt ordinary method carrier construction E; Synthetic be used to the to increase primer of tissue-specific promoter; Genomic dna with human archeocyte is that template is carried out pcr amplification; The PCR product that must have tissue-specific promoter's sequence; The PCR product that will have tissue-specific promoter's sequence with and the restriction enzyme I of tissue-specific promoter's MCS of carrier E and corresponding restriction enzyme I of restriction enzyme site and the II double digestion of II; Be inserted into tissue-specific promoter's MCS of carrier E, adopt ordinary method to make up two heterogeneic carrier F that contain the dual-gene reticent shRNA expression cassette of tissue specificity of target;
Or the MCS place that in support C, inserts tissue-specific promoter inserts earlier through pcr amplification; Tissue-specific promoter behind the double digestion; Adopt ordinary method carrier construction G; The MCS place that in carrier G first inserts the shRNA template sequence inserts synthetic again after first shRNA template sequence of double digestion; Adopt ordinary method carrier construction H, second MCS place that inserts the shRNA template sequence in carrier H inserts synthetic after second shRNA template sequence of double digestion adopts ordinary method to make up two heterogeneic carrier R that contain the dual-gene reticent shRNA expression cassette of tissue specificity of target.
4. the application that comprises from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site according to claim 3; Comprise that the carrier F that contains the dual-gene reticent shRNA expression cassette of tissue specificity or the dual-gene reticent shRNA expression cassette of the tissue specificity among the carrier R that will make up are inserted among other different carrier K, adopt ordinary method to make up different containing from the dual-gene reticent RNAi expression vector H of the tissue specificity of shearing site; Described carrier K is for inserting the exogenous nucleic acid fragment, can carrying exogenous nucleic acid and get into mammalian cell and the nucleic acid molecule that carries out independent and stable self-replacation therein.
5. the application that comprises from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site according to claim 3 is characterized in that, is realized by following steps:
(1) first has from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert initial carrier pEGFP-C1 for ease; 5 ' the terminal Nhe I restriction enzyme site GCTAGC that adds at first cis acting Hammerhead ribozyme sequence SEQ ID NO:1; At 3 ' the terminal Bgl II restriction enzyme site AGATCT that adds; Synthetic, an oligonucleotide with cis acting Hammerhead ribozyme sequence of winning is double-stranded, with restriction enzyme Nhe I and Bgl II double digestion; Insert Nhe I and the Bgl II restriction enzyme site of initial carrier pEGFP-C1, adopt ordinary method carrier construction pRib SEQ ID NO:7 thus;
(2) second have from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert carrier pRib for ease; 5 ' the terminal HindIII restriction enzyme site AAGCTT that adds at second cis acting Hammerhead ribozyme sequence SEQ ID NO:1; 3 ' the terminal EcoR I restriction enzyme site GAATTC that adds; Synthetic, it is double-stranded to get second oligonucleotide with cis acting Hammerhead ribozyme sequence, with restriction enzyme HindIII and EcoR I double digestion; First that is inserted into carrier pRib adopts ordinary method carrier construction pDi-Rib SEQ ID NO:8 thus from shearing the HindIII and the EcoR I restriction enzyme site in ribozyme sequence downstream;
(3) have the insertion of the oligonucleotide of transcription termination sequence:
Insert carrier pDi-Rib for ease; 5 ' the terminal Kpn I restriction enzyme site GGTACC that adds at zinc finger protein MAZ binding sequence SEQ ID NO:2; 3 ' the terminal BamH I restriction enzyme site GGATCC that adds; Synthetic; The oligonucleotide two strands that must have zinc finger protein MAZ binding sequence is sheared the Kpn I and the BamH I restriction enzyme site in ribozyme sequence downstream certainly, is adopted ordinary method carrier construction pTSDS SEQ ID NO:9 thus for second that is inserted into carrier pDi-Rib with restriction enzyme Kpn I and BamH I double digestion;
(4) first shRNA template sequence MCS inserts hTERT shRNA sequence:
Insert carrier pTSDS for ease; 5 ' the terminal Bgl II restriction enzyme site that adds at hTERT shRNA sequence SEQ ID NO:3; 3 ' the terminal HindIII restriction enzyme site that adds, synthetic, the oligonucleotide that must contain hTERT shRNA sequence is double-stranded; With restriction enzyme Bgl II and HindIII double digestion; Be inserted into the MCS of first shRNA template sequence of carrier pTSDS after enzyme is cut, promptly Bgl II and HindIII restriction enzyme site adopt ordinary method to make up the carrier pTSDS-t SEQID NO:10 that obtains target hTERT thus;
(5) second shRNA template sequence MCSs insert JunB shRNA sequence:
Insert carrier pTSDS-t for ease; 5 ' the terminal EcoR I restriction enzyme site that adds at JunB shRNA sequence SEQ ID NO:4; 3 ' the terminal Kpn I restriction enzyme site that adds, synthetic, the oligonucleotide that must contain JunB shRNA sequence is double-stranded; With restriction enzyme EcoR I and Kpn I double digestion; Be inserted into the MCS of second shRNA template sequence of carrier pTSDS-t after enzyme is cut, promptly EcoR I and Kpn I restriction enzyme site adopt ordinary method to make up the carrier pTSDS-tb SEQ ID NO:11 that obtains target hTERT and JunB thus;
(6) amplification of apoA-I promotor, and apoA-I promotor is inserted tissue-specific promoter's MCS:
Synthetic be used to the to increase primer of apoA-I promotor: upstream primer 5 '-ATTAATaggggaaggggatgagtg-3 '; Downstream primer 5 '-GCTAGCgagaagaagggcctggctga-3 '; With human liver cancer cell SMMC-7721 genome is that template is carried out pcr amplification; Obtain having the PCR product of apoA-I promoter sequence SEQ ID NO:24; The PCR product that will have the apoA-I promoter sequence is inserted into tissue-specific promoter's MCS of carrier pTSDS-tb with restriction enzyme A se I and Nhe I double digestion, i.e. Ase I and Nhe I restriction enzyme site; Through sequence verification, adopt ordinary method to make up and obtain target hTERT and the dual-gene specific dual-gene reticent RNAi expression vector pTSDS/apo-tb SEQ ID NO:12 of hepatic tissue of JunB simultaneously.
6. the application that comprises from the dual-gene reticent RNAi expression vector of the tissue specificity of shearing site according to claim 3 is characterized in that, is realized by following steps:
(1) first has from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert initial carrier pEGFP-C1 for ease; 5 ' the terminal Nhe I restriction enzyme site GCTAGC that adds at first cis acting Hammerhead ribozyme sequence SEQ ID NO:1; At 3 ' the terminal Bgl II restriction enzyme site AGATCT that adds; Synthetic, an oligonucleotide with cis acting Hammerhead ribozyme sequence of winning is double-stranded, with restriction enzyme Nhe I and Bgl II double digestion; Insert Nhe I and the Bgl II restriction enzyme site of initial carrier pEGFP-C1, adopt ordinary method carrier construction pRib SEQ ID NO:7 thus;
(2) second have from the insertion of shearing the oligonucleotide of ribozyme sequence:
Insert carrier pRib for ease; 5 ' the terminal HindIII restriction enzyme site AAGCTT that adds at second cis acting Hammerhead ribozyme sequence SEQ ID NO:1; 3 ' the terminal EcoR I restriction enzyme site GAATTC that adds; Synthetic, it is double-stranded to get second oligonucleotide with cis acting Hammerhead ribozyme sequence, with restriction enzyme HindIII and EcoR I double digestion; First that is inserted into carrier pRib adopts ordinary method carrier construction pDi-Rib SEQ ID NO:8 thus from shearing the HindIII and the EcoR I restriction enzyme site in ribozyme sequence downstream;
(3) have the insertion of the oligonucleotide of transcription termination sequence:
Insert carrier pDi-Rib for ease; 5 ' the terminal Kpn I restriction enzyme site GGTACC that adds at zinc finger protein MAZ binding sequence SEQ ID NO:2; 3 ' the terminal BamH I restriction enzyme site GGATCC that adds; Synthetic; The oligonucleotide two strands that must have zinc finger protein MAZ binding sequence is sheared the Kpn I and the BamH I restriction enzyme site in ribozyme sequence downstream certainly, is adopted ordinary method carrier construction pTSDS SEQ ID NO:9 thus for second that is inserted into carrier pDi-Rib with restriction enzyme Kpn I and BamH I double digestion;
(4) amplification of hTERT promotor, and hTERT promotor is inserted tissue-specific promoter's MCS:
Synthetic be used to the to increase primer of hTERT promotor: upstream primer 5 '-ATTAATtccgcccggagcagctgcg-3 '; Downstream primer; 5 '-GCTAGCgcagcactcgggccaccag-3 '; With human liver cancer cell SMMC-7721 genome is that template is carried out pcr amplification, obtains having the PCR product of hTERT promoter sequence SEQ ID NO:25, with restriction enzyme A se I and Nhe I double digestion; After cutting, enzyme is inserted into tissue-specific promoter's MCS of carrier pTSDS; Be Ase I and Nhe I restriction enzyme site,, adopt ordinary method to make up the dual-gene reticent RNAi expression vector pTSDS/tert SEQ ID NO:13 that obtains having tumour-specific through sequence verification;
(5) first shRNA template sequence MCS inserts hEGFR shRNA sequence:
Insert carrier pTSDS/tert for ease; 5 ' the terminal Bgl II restriction enzyme site that adds at hEGFR shRNA sequence SEQ ID NO:5; 3 ' the terminal HindIII restriction enzyme site that adds, synthetic, the oligonucleotide that must contain hEGFR shRNA sequence is double-stranded; With restriction enzyme Bgl II and HindIII double digestion; Be inserted into the MCS of first insertion shRNA template sequence of carrier pTSDS/tert after enzyme is cut, promptly Bgl II and HindIII restriction enzyme site adopt ordinary method to make up the carrier pTSDS/tert-e SEQ ID NO:14 that obtains target hEGFR thus;
(6) second shRNA template sequence MCSs insert c-fos shRNA sequence:
Insert carrier pTSDS/tert-e for ease; 5 ' the terminal EcoR I restriction enzyme site that adds at c-fos shRNA sequence SEQ ID NO:6; 3 ' the terminal Kpn I restriction enzyme site that adds; Synthetic, the oligonucleotide that must contain c-fos shRNA sequence is double-stranded, with restriction enzyme EcoR I and Kpn I double digestion; Be inserted into EcoR I and pn I restriction enzyme site in the MCS of second shRNA template sequence of carrier pTSDS/tert-e after enzyme is cut, adopt ordinary method to make up to obtain the dual-gene silent carrier pTSDS/tert-ef of the tumor tissues specificity SEQ ID NO:15 of target c-fos and hEGFR simultaneously;
(7) structure of the dual-gene silencing virus carrier of tumor tissues specificity:
With the dual-gene silent carrier pTSDS/tert-ef of the tumour-specific of while target c-fos and hEGFR with restriction enzyme A se I and BamH I double digestion, and with T 4Archaeal dna polymerase is mended sticky end flat, and agarose gel electrophoresis reclaims small segment SEQ ID NO:17, with restriction enzyme EcoR I and HindIII double digestion retroviral vector pRS, and with T 4Archaeal dna polymerase is mended sticky end flat, and agarose gel electrophoresis reclaims big fragment SEQ ID NO:18, with T 4Dna ligase is connected above-mentioned small segment with big fragment, adopt ordinary method to make up and obtain the dual-gene silencing virus carrier of tumor tissues specificity pTSDS/MMLV/tert-ef SEQ ID NO:16.
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