CN104805120A - ShRNA-Ago2 coexpression lentivirus RNAi vector, recombinant plasmid and constructing method of recombinant plasmid - Google Patents

Abstract

The invention discloses an shRNA-Ago2 coexpression lentivirus RNAi vector, a recombinant plasmid and a constructing method of the recombinant plasmid. The shRNA-Ago2 coexpression lentivirus RNAi vector includes an shRNA expression cassette and an Ago2 protein expression cassette, and the shRNA expression cassette includes a promoter U6, H1 or 7SK, and a ccdB lethal gene; and the Ago2 protein expression cassette includes a CMV strong promoter, an Ago2 protein expression gene, an internal ribosome entry site DNA sequence and a ZsGreen1 green fluorescin expression gene. After the vector is introduced into cells, shRNA for interfering the target gene and the enhancement factor Ago2 and ZsGreen1 green fluorescin report gene can be simultaneously expressed. The vector co-expresses Ago2 to substantially enhance the silencing effect of shRNA, the operation is simple and rapid, the effect and the lasting time of the RNAi interference gene are greatly improved, and the vector can be used to treat tumors and other serious diseases.

Description

A kind of shRNA-Ago2 coexpression slow virus RNAi carrier, recombinant plasmid and construction process thereof

Technical field

The invention belongs to functional genomics research field, relate to a kind of slow virus RNAi carrier, recombinant plasmid and construction process thereof.

Background technology

RNA disturbs (RNA interference, RNAi) be a kind of high conservative during evolution, by double-stranded RNA (double-stranded RNA, dsRNA) silencing phenomenon (Fire A. after the genetic transcription of the efficient selective degradation of homologous mRNA is brought out, Nature, 1998.391 (6669): p.806-11.).RNAi technology does not only disclose genes within cells Silencing Mechanisms, is effective research means of the another deciphering gene function after the technology such as yeast crossbreeding system, DNA chip, gene knockout, sense-rna, greatly facilitates the process that the mankind disclose life secret.The mechanism of action of RNA interference in cell is divided into three phases: first is initial period, exogenous or endogenous double stranded rna molecule is cut into the small molecules double-strand (small-interfering RNAs, siRNA) of 21-25 length of nucleotides by intracellular Dicer enzyme processing; Second is assembling stage of RISC, wherein a chain and Argonaute(Ago in the siRNA of generation) and associated protein assemble form tool activated silencing complex RISC(RNA-induced silencingcomplex); 3rd is the effective stage, the activated RISC of tool that assembling is formed acts on the mRNA with the strand microRNA complementary on it, be suppressed by making it translate this mRNA degraded, finally cause the silence (Siomi of gene, H.and M.C.Siomi, Nature, 2009.457 (7228): p.396-404).

But, often find in the practical application of RNAi, even the shRNA of appropriate design still can not play effective interference effect.By analysis, summarized several factors affecting shRNA interference effect: as drive siRNA to express promotor, siRNA action site, the tandem expression of multiple siRNA, siRNA and RISC the (Dykxhoorn such as the transfection efficiency in conjunction with situation and interference carrier, D.M.and J.Lieberman, Annu Rev Biomed Eng, 2006.8:p.377-402).

For the problems referred to above, researchist is devoted to the RNAi associated protein factor finding to strengthen shRNA jamming effectiveness.Ago2 is the protein uniquely including nicking activity in Argonaute protein family.Intracellular rna i be unable to do without participation (the JuvvunaP.K..Nucleic Acids Res of Ago2,2012.40 (14): p.6808-20), Ago2 not only can be combined with siRNA, also by being combined with miRNA thus improving the level of ripe miRNA in cell and stability (Ghildiyal M.and P.D.Zamore.Nat Rev Genet, 2009.10 (2): p.94-108).Intracellular siRNA and miRNA is then combined with limited amount Ago2 competitively, external source imports siRNA may cause following situation: on the one hand, in cell, most of Ago2 may be occupied by miRNA, the shRNA that external source imports then may load it owing to not having enough Ago2, thus causes part shRNA to fail to play a role and be just degraded; On the other hand, research shows that siRNA, shRNA exist advantage than pre-miRNA in conjunction with in RISC, illustrate that the external source shRNA entering cell more preferentially than miRNA may take RISC in cell and namely consume Ago2 (Castanotto D.Nucleic Acids Res, 2007.35 (15): p.5154-64), so endogenous miRNA may be degraded owing to not having enough Ago2 to load it, causes the cellular activity relying on miRNA approach to be affected.Therefore, in cell, the quantity available of Ago2 is not enough, not only can affect the performance of shRNA interference effect, also may affect the running of miRNA normal function in cell.Under the prerequisite maintaining cell miRNA normal function, the efficiency of intracellular rna i depends on the quantity of available Ago2 to a great extent.There are some researches prove, process LAN Ago2 albumen can improve the RNAi efficiency in mammalian cell.

Diederichs etc. pass through Ago2 and shRNA cotransfection in HEK293 cell, the luciferase expression amount detected in cell finds that Ago2 can significantly improve RNAi silence efficiency (Diederichs S.Proc Natl Acad Sci U S A, 2008.105 (27): p.9284-9).Grimm etc. are co expression shRNA and Ago2 in mouse, find that Ago2 can obviously strengthen silence efficiency and extend quiet hour, toxicity (Grimm, D.J Clin Invest, 2010.120 (9): p.3106-19) that shRNA brings to mouse liver can be reduced simultaneously.But, adopt the mode of Ago2 and shRNA cotransfection import to cell be difficult to control both between ratio.For this problem, shRNA and Ago2 is structured on same expression vector by Chen etc., and expression vector is imported in African toad neurocyte and carry out RNAi experiment, result shows that this expression vector has higher interference level (Chen than common shRNA in neurocyte, C.M.Front Neurosci, 2009.3:p.63.).But the transfection efficiency of shRNA expression vector in cell is not high in above-mentioned experiment, have impact on the high level expression of shRNA at cell; Meanwhile, above-mentioned technology is all the transient expression (namely the short period of time expresses) carrying out carrier, is difficult to the long duration of action maintaining shRNA; In addition, the condition non-optimal such as the kind of genetic expression functional element that prior art adopts, structure and array mode, this also counteracts that effective performance of shRNA interference effect.Therefore, the technology of existing expression shRNA has yet to be improved and developed.

Summary of the invention

In order to solve the problem, the invention provides a kind of shRNA-Ago2 coexpression slow virus RNAi carrier, recombinant plasmid and construction process thereof, sustainable after introducing cell, efficient shRNA and the enhancement factor Ago2 simultaneously expressing goal gene of this carrier.

To achieve these goals, the present invention adopts following technical scheme:

A kind of shRNA-Ago2 coexpression slow virus RNAi carrier, comprise shRNA expression cassette and Ago2 protein expression frame, described shRNA expression cassette comprises promotor U6, H1 or 7SK, and ccdB lethal gene; Described Ago2 protein expression frame comprises CMV strong promoter, Ago2 protein-encoding gene, internal ribosome entry site DNA sequence dna and ZsGreen1 egfp expression gene.

Preferably, the two ends of described ccdB lethal gene are provided with restriction enzyme site BamHI and MluI.

A construction process for shRNA-Ago2 coexpression slow virus RNAi carrier, comprises the following steps:

(1) for the preparation of the skeleton pLVX-IRES-ZsGreen1-Puro of carrier construction:

With pLVX-IRES-ZsGreen1 carrier for template, amplification obtains IRES-ZsGreen1 fragment, by Cla1-Xba1 double digestion PCR primer, is then cloned into pLVX-Puro carrier by corresponding site, obtains pLVX-IRES-ZsGreen1-Puro carrier framework.

(2) enhancement factor Ago2 gene expression frame CMV-Ago2 is inserted:

With MluI and EcoRI double digestion pIRESneo-FLAG/HA-Ago2 carrier, obtain CMV-Ago2 fragment, the pLVX-IRES-ZsGreen1-Puro carrier framework that CMV-Ago2 fragment and step (1) reclaim is connected, proceed to competent escherichia coli cell, be coated with LB dull and stereotyped, overnight incubation, carries out bacterium colony PCR and enzyme cuts qualification, the carrier pLVX-CMV-Ago2-IRES-ZsGreen1-Puro obtained;

(3) insert shRNA and express promotor:

With the carrier pLVX-CMV-Ago2-IRES-ZsGreen1-Puro that BclI and MluI double digestion step (2) obtains, dephosphorization acid rear electrophoresis reclaims; After PCR primer by the amplification of BclI and MluI double digestion hU6, H1 or 7SK promotor, be connected on the corresponding site of pLVX-CMV-Ago2-IRES-ZsGreen1-Puro carrier, proceed in competent escherichia coli cell, be coated with LB dull and stereotyped, overnight incubation, the carrier obtained after identifying is: pLVX-hU6/H1/7SK-CMV-Ago2-IRES-ZsGreen1-Puro.

(4) insert ccdB sequence and obtain shRNA-Ago2 coexpression slow virus RNAi carrier:

With pLenti6 carrier for template, amplify the fragment containing intestinal bacteria ccdB lethal gene, after the ccdB BamHI arrived of amplification and Mlu I double digestion, be connected with the pLVX-hU6/H1/7SK-CMV-Ago2-IRES-ZsGreen1-Puro carrier through BamHI and Mlu I double digestion, proceed in competent escherichia coli cell, be coated with the dual anti-flat board of LB/amp+Chl, overnight incubation, carry out bacterium colony PCR and enzyme cuts qualification, obtain shRNA-Ago2 coexpression slow virus RNAi carrier, carrier framework is pLVX-hU6/H1/7SK-ccdB-CMV-Ago2-IRES-ZsGreen1-Puro.

In such scheme, can use the expression of hU6, H1 or 7SK promoters driven shRNA on the same vector, CMV promoter drives the expression of Ago2 and the expression of green fluorescent protein simultaneously.CcdB two ends are provided with restriction enzyme site BamHI and MluI, for building and screen the shRNA-Ago2 coexpression slow virus RNAi carrier containing goal gene shRNA positive colony.The expression of enhancement factor Ago2 was loaded into silencing complex RISC with both having ensure that the shRNA maximum of transfered cell, was convenient to the silence that shRNA sheared to its goal gene, strengthen goal gene; Simultaneously also for maintaining normally carrying out of the RNAi path of miRNA mediation in cell, reduce the impact that external source imports shRNA cellular function.

Present invention also offers a kind of shRNA-Ago2 coexpression slow virus recombinant plasmid, described shRNA-Ago2 coexpression slow virus recombinant plasmid comprises above-mentioned shRNA-Ago2 coexpression slow virus RNAi carrier.

Preferably, described shRNA-Ago2 coexpression slow virus recombinant plasmid also comprises goal gene shRNA, and stem's length of described shRNA is 19 ~ 22bp, and the loop length of described shRNA is 4 ~ 9bp.

Preferably, stem's length of described shRNA is 20bp.

Preferably, the loop length of described shRNA is 6 ~ 9bp.

Preferably, the sense/antisense structure of described shRNA is the sense-loop-antisense structure from 5 ' end.

Present invention also offers a kind of construction process of shRNA-Ago2 coexpression slow virus recombinant plasmid, comprise the following steps:

1) for goal gene design shRNA encoding sequence, two primer annealings are formed the shRNA of band restriction enzyme BamHI and MluI cohesive terminus by chemosynthesis two primers;

2) digest above-mentioned shRNA-Ago2 coexpression slow virus RNAi carrier skeleton by restriction enzyme BamHI and MluI, after enzyme cuts back to close, carrier framework is connected with the shRNA of annealing, transformation of E. coli competent cell coated plate overnight incubation;

3) the mono-clonal bacterium colony that flat board grows is carried out DNA sequencing, determine the correct insertion of shRNA encoding sequence, obtain shRNA-Ago2 coexpression slow virus recombinant plasmid.

In such scheme, the present invention utilizes the characteristic of lethal gene ccdB in escherichia coli expression, the shRNA of goal gene is inserted shRNA-Ago2 coexpression slow virus RNAi carrier, effectively can remove template and pollute, accomplish Zero background constructing rna interference vector.The building process of slow virus RNAi carrier of the present invention is simply efficient, and those skilled in the art can use this carrier Effective selection to contain the positive colony of interference goal gene shRNA Insert Fragment, rapid build shRNA library.

ShRNA-Ago2 coexpression slow virus RNAi carrier provided by the invention is based on slow virus expression system.Lot of documents research shows, goal gene is also incorporated in Unseparated Cell by lentiviral vectors energy productive infection cell, and the tissue that lentiviral vectors is applicable to or cell type comprise brain, liver, muscle, retina, hemopoietic stem cell, mesenchymal stem cells MSCs, scavenger cell etc.This characteristic makes lentiviral vectors compared with other virus vector, as adenovirus carrier (unconformability genome), gland relevant viral vector (integration rate is low), traditional retroviral vector (only integrating somatoblast), has distinct advantage.Use the cell of plasmid transient transfection compared to other, the efficiency of slow virus infected cell is higher.Therefore, slow virus RNAi carrier provided by the invention is used to may be used for building clone that is stable, coexpression goal gene shRNA and Ago2 constantly.

Compared with prior art, the present invention has clear superiority:

1. the present invention optimizes through comprehensive in structure design for the shRNA of carrier construction, pass through optimum combination, the kind of the Expression element involved by this carrier, structure and array mode all have obvious enhancing for shRNA silencing efficiency, reach the object improving RNA silence efficiency.ShRNA-Ago2 coexpression slow virus RNAi carrier function of the present invention is excellent and easy to use, high to the infection rate of higher eucaryotic cells, to the reticent effect lasts of goal gene, efficiently, facilitates investigator to apply RNAi technology better and conducts a research.

2. the present invention uses same lentiviral vectors coexpression goal gene shRNA and enhancement factor Ago2, this carrier can express shRNA and the enhancement factor Ago2 of goal gene simultaneously after introducing cell, the silence effect of shRNA is significantly strengthened by coexpression Ago2, this expression system not only significantly improve shRNA silencing efficiency, ensure that the cell function that microRNA approach mediates normally runs, also a saving cost and the time of vector construction and screening.Owing to only needing a kind of plasmid of transfection in cell transfecting process, therefore improve cell transfecting efficiency and reduce screening process.Utilize present method to disturb the experimental period of goal gene short, transfection efficiency is high, and shRNA silencing efficiency is remarkable.

3. the present invention builds shRNA-Ago2 coexpression slow virus RNAi carrier based on slow virus expression system, has the advantage that infection rate is high, the interference effect time length is long, integration ability is strong and ability to express is high.Compared to the transient transfection cell technology based on plasmid vector, RNAi silence efficiency is no longer subject to the restriction of cell fission, transfection efficiency or expression level.

4. the present invention builds shRNA-Ago2 coexpression slow virus RNAi carrier based on slow virus expression system, containing ZsGreen1 reporter gene, can detect packaged virus titer and cell transduction efficiency flexibly and easily by ZsGreen1 reporter gene.

Accompanying drawing explanation

Fig. 1 is embodiment of the present invention 1-3 slow virus RNAi carrier collection of illustrative plates.

Fig. 2 be in the embodiment of the present invention 6 shRNA-Ago2 coexpression slow virus RNAi carrier to the silencing efficiency figure of foreign gene DsRed.

Fig. 3 be in the embodiment of the present invention 7 shRNA-Ago2 coexpression slow virus RNAi carrier to the silencing efficiency figure of endogenous gene I/D 1.

Fig. 4 is that stem's length of shRNA in the embodiment of the present invention 8 is to the effect diagram of RNAi.ShRNA stem length 19 ~ 22nt all have more than 70% silencing efficiency.

Fig. 5 is that the loop length of shRNA in the embodiment of the present invention 8 and sense/antisense structure are to the effect diagram of RNAi.Loop length is the shRNA that the silencing efficiency in the shRNA of 6 ~ 9nt is better than that loop length is 4nt, and structure is that the shRNA of sense-antisense is stronger than the silencing efficiency of antisense-sense.

Fig. 6 be in the embodiment of the present invention 9 different promoters to the effect diagram of RNAi.

Embodiment

The invention provides a kind of slow virus RNAi carrier of shRNA-Ago2 coexpression, recombinant plasmid and construction process thereof, for making object of the present invention, technical scheme and effect clearly, clearly, the present invention is described in more detail below.Except specified otherwise, method, reagent etc. that the present invention uses are all well-known to those skilled in the art, do not repeat them here.Specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.

The embodiment of the present invention 1 ~ 3 passes through molecular biology method, successively the elements such as shRNA promotor, ccdB lethal gene CMV strong promoter, Ago2 protein-encoding gene, IRES gene order and green fluorescent protein (ZsGreen1) expressing gene are loaded into slow virus skeleton, structure obtains shRNA-Ago2 coexpression slow virus RNAi carrier of the present invention, by its called after pLVE-shRNA-Ago2, Vector map is shown in Fig. 1.

The present invention used experiment material source in table 1.

Table 1 experiment material and source

The structure of embodiment 1shRNA-Ago2 coexpression slow virus RNAi carrier

(1) for the preparation of the skeleton pLVX-IRES-ZsGreen1-Puro of carrier construction:

With pLVX-IRES-ZsGreen1 (Clontech) carrier for template, by upstream primer (SEQ ID No:1) TTTATCGATGGATCCTAACGCGTGAATTCGCCCCTCTCCCTC and downstream primer (SEQ ID No:2) GGTCTAGATCAGGGCAAGGCGGAGC amplification IRES-ZsGreen1 fragment, by Cla1-Xba1 double digestion PCR primer, then be cloned into pLVX-Puro carrier (Clontech) by corresponding site, obtain pLVX-IRES-ZsGreen1-Puro carrier framework.

(2) enhancement factor Ago2 gene expression frame CMV-Ago2 is inserted:

With MluI and EcoRI double digestion pIRESneo-FLAG/HA-Ago2 carrier (purchased from Invitrogen, containing CMV promoter and enhancement factor Ago2 sequence), obtain the CMV-Ago2 fragment of about 3.3kb, the pLVX-IRES-ZsGreen1-Puro carrier framework of CMV-Ago2 fragment with step (1) is connected by corresponding restriction enzyme site, proceed to competent escherichia coli cell stable3, be coated with the dull and stereotyped 37 DEG C of overnight incubation of LB, carry out bacterium colony PCR and enzyme cuts qualification, the carrier pLVX-CMV-Ago2-IRES-ZsGreen1-Puro obtained;

(3) insert shRNA and express promotor

Use hU6 promotor in the present embodiment.With the carrier pLVX-CMV-Ago2-IRES-ZsGreen1-Puro that BclI and MluI double digestion step (2) obtains, dephosphorization acid rear electrophoresis reclaims; Be designed for the primer (SEQ ID No:5-6) of amplification hU6 promotor, introduce restriction enzyme site BclI and MluI, be that template amplification obtains hU6 promotor with human genome DNA, after the PCR primer BclI obtained and MluI double digestion, be connected on the corresponding site of pLVX-CMV-Ago2-IRES-ZsGreen1-Puro carrier, proceed in intestinal bacteria stable3 competent cell, be coated with the dull and stereotyped 37 DEG C of overnight incubation of LB, the carrier obtained after identifying is: pLVX-hU6-CMV-Ago2-IRES-ZsGreen1-Puro.

(4) insert ccdB sequence and obtain complete shRNA-Ago2 coexpression slow virus RNAi carrier:

With upstream primer (SEQ ID No:3)

CGAGGATCCGAATTCTGCAGATATCAACAAGTTTGTACAAAAAAG and downstream primer (SEQ ID No:4) TCAACGCGTCAAGTCGAGCGGCCGCCACTGTGC is from the fragment of pLenti6 carrier (Invitrogen) amplification containing intestinal bacteria ccdB lethal gene, be connected with the pLVX-hU6-CMV-Ago2-IRES-ZsGreen1-Puro carrier of BamHI and Mlu I double digestion with after BamHI with Mlu I double digestion, proceed in competent escherichia coli cell DB3.1, be coated with the dual anti-flat board of LB/amp+Chl, 37 DEG C of overnight incubation, carry out bacterium colony PCR and enzyme cuts qualification, obtain shRNA-Ago2 coexpression slow virus RNAi carrier, carrier framework is pLVX-hU6-ccdB-CMV-Ago2-IRES-ZsGreen1-Puro.

Table 2 is for the Oligonucleolide primers of the promotor that increases

The structure of embodiment 2shRNA-Ago2 coexpression slow virus RNAi carrier

(1) for the preparation of the skeleton pLVX-IRES-ZsGreen1-Puro of carrier construction: with embodiment 1;

(2) enhancement factor Ago2 gene expression frame CMV-Ago2 is inserted: with embodiment 1;

(3) insert shRNA and express promotor

Use H1 promotor in the present embodiment, primer sequence is SEQ ID No:7-8 (see table 2).The carrier obtained with reference to the preparation method of embodiment 1 is: pLVX-H1-CMV-Ago2-IRES-ZsGreen1-Puro.

(4) insert ccdB sequence and obtain complete shRNA-Ago2 coexpression slow virus RNAi carrier:

With embodiment 1, obtain the shRNA-Ago2 coexpression slow virus RNAi carrier that carrier framework is pLVX-H1-ccdB-CMV-Ago2-IRES-ZsGreen1-Puro.

The structure of embodiment 3shRNA-Ago2 coexpression slow virus RNAi carrier

With reference to embodiment 1 and 2, the present invention also can use 7SK promotor, primer sequence is SEQ ID No:9-10 (see table 2), prepares the shRNA-Ago2 coexpression slow virus RNAi carrier that carrier framework is pLVX-7SK-ccdB-CMV-Ago2-IRES-ZsGreen1-Puro.

The shRNA-Ago2 coexpression slow virus recombinant plasmid of embodiment 4 external source goal gene and construction process thereof

For the shDsRed-23 of reticent red fluorescent protein, shRNA-Ago2 coexpression slow virus recombinant plasmid and the construction process thereof of reticent goal gene is described:

1) for goal gene design shRNA encoding sequence, synthesize shDsRed-23 oligonucleotide primer sequence (see table 3) respectively, mixed, be positioned in boiling water and be naturally down to annealing at room temperature formation double-strand shDsRed-23, double-strand shDsRed-23 two ends restricted property restriction endonuclease BamHI and MluI cohesive terminus respectively; With the primer shown in the shcontrol in table 3 for contrast;

2) the shRNA-Ago2 coexpression slow virus RNAi carrier in any one of embodiment 1-3 is digested by restriction enzyme BamHI and MluI, after enzyme cuts back to close, it is connected with the shDsRed-23 synthesized that anneals, use intestinal bacteria stable3 competent cell to transform, be coated with the dull and stereotyped 37 DEG C of overnight incubation of LB;

3) the mono-clonal bacterium colony that flat board grows is carried out DNA sequencing, determine the correct insertion of shRNA encoding sequence, obtain shRNA-Ago2 coexpression slow virus recombinant plasmid.

The primer sequence of reference aforesaid method and table 3, can build the shRNA-Ago2 coexpression slow virus recombinant plasmid of reticent other positions of shDsRed gene.

The shRNA oligonucleotide sequence of table 3 target DsRed gene coding region different positions

The pLVE-shRNA-Ago2 coexpression slow virus recombinant plasmid of the endogenous goal gene of embodiment 5 and construction process thereof

For reticent differentiation inhibiting factor ID1-285 gene, shRNA-Ago2 coexpression slow virus RNAi carrier and the construction process thereof of silencing endogenous goal gene is described:

1) for goal gene design shRNA encoding sequence, synthesize shID1-285 oligonucleotide primer sequence (see table 4) respectively, mixed, be positioned in boiling water and be naturally down to annealing at room temperature formation double-strand shID1-285, double-strand shID1-285 two ends restricted property restriction endonuclease BamHI and MluI cohesive terminus respectively; The contrast of gene shRNA for the purpose of shcontrol;

2) the shRNA-Ago2 coexpression slow virus RNAi carrier in any one of embodiment 1-3 is digested by restriction enzyme BamHI and MluI, after enzyme cuts back to close, it is connected with the sh ID1-285 synthesized that anneals, use intestinal bacteria stable3 competent cell to transform, be coated with the dull and stereotyped 37 DEG C of overnight incubation of LB;

3) the mono-clonal bacterium colony that flat board grows is carried out DNA sequencing, determine the correct insertion of shRNA encoding sequence, obtain shRNA-Ago2 coexpression slow virus recombinant plasmid.

The primer sequence of reference aforesaid method and table 4, can build the shRNA-Ago2 coexpression slow virus recombinant plasmid of reticent other positions of shID1 gene.

The shRNA oligonucleotide sequence of table 4 target ID1 gene coding region different positions

Embodiment 6shRNA-Ago2 coexpression slow virus RNAi carrier is to the silencing efficiency of foreign gene

The shRNA-Ago2 coexpression slow virus recombinant plasmid prepared for embodiment 4 carries out shRNA-Ago2 coexpression slow virus RNAi carrier to the experiment of the silencing efficiency of goal gene, specifically comprises the following steps:

The slow virus packaging of 1.shRNA-Ago2 coexpression slow virus RNAi carrier:

(1.1) use the nutrient solution without tsiklomitsin to cultivate 293T cell, transfectional cell inoculates 293T cell in 100mm plate (10ml is without tsiklomitsin DMEM) the day before yesterday, cultivates 12 ~ 24h.

(1.2) treat that the density of cell reaches 80-90%, the plasmid mixed solution that this rotaring redyeing system often organizes needs is 17.1 μ g packaging plasmids (Addgene company) and 6.9 μ g pLVE-shRNA-Ago2 coexpression slow virus recombinant plasmids, after fully being mixed by above-mentioned plasmid, add NaCl(150mM) make final volume be 500 μ l; Often group needs transfection reagent 63 μ l PEI+437 μ l NaCl(150mM), final volume is 500 μ l.Test with the shRNA Lentiviral not containing Ago2 gene as contrast.

(1.3) join in DNA solution by transfection reagent, mix, room temperature leaves standstill 10min.

(1.4) be that the mixed solution of 1mL is dropwise slowly added drop-wise in the culture dish of 100mm by volume, after 8-12h cultivated by 37 DEG C of incubators, renew fresh in tsiklomitsin nutrient solution; In order to improve the concentration of virus, when changing liquid, 8ml nutrient solution can be changed into.

(1.5) collect supernatant after cultivating 48h again, change into fresh without tsiklomitsin nutrient solution in culture dish.The centrifugal 10min of 3500rpm, the supernatant liquor of collection, i.e. corresponding virus liquid.

(1.6) often manage in the EP pipe of average packing about 700 μ l to 1.5ml sterilizing.Same method process after 72h, collects vial supernatant.

2.shRNA-Ago2 coexpression slow-virus transfection Hela clone:

(2.1) by Hela cell kind in 6 orifice plates, cell density is 1.5 × 10 ⁵individual cells/well.

(2.2), after cultivating 12 ~ 24h, with 500 μ l DMEM+500 μ l shRNA-Ago2 coexpression slow virus liquid+0.5 μ l polybrene (polybrene, final concentration is 10 μ g/ μ l) nutrient solutions, virus infection is carried out to Hela cell.With the slow virus of the shRNA Lentiviral not containing Ago2 gene for contrast.

(2.3) fresh medium is changed after infecting viral 24h.

(2.4) after cell infection virus 72h, observe fluorescence, and add tetracycline (puromycin) the nutrient solution continuation culturing cell that final concentration is 1 μ g/ μ l, until filter out the clone of stable transfection shRNA-Ago2 coexpression slow virus.

3. flow cytomery cell fluorescence expression amount:

(3.1) first discard supernatant liquor in hole, clean cell 2 times with PBS.

(3.2) with 0.25% trypsin digestion cell of 100 μ l, then the DMEM adding 200 μ l dispels cell.

(3.3) by cell harvesting in 1.5ml EP pipe, after the centrifugal 2min of 1000rpm, supernatant discarded.

(3.4) 1ml PBS re-suspended cell is added, by luciferase expression amount in flow cytomery cell.

(3.5) data analysis:

The expression amount of two kinds of fluorescence (EGFP, DsRed) in flow cytomery cell.Data Processing in Experiment is as follows: F=[(A-B)/(C-G)]/[(D-B)/(E-G)], wherein F is for being normalized experimental group fluorescence relative expression quantity with control group, A, C is experimental group two kinds of fluorescence (EGFP, DsRed) expression amount, D, E is control group two kinds of fluorescence (EGFP, DsRed) expression amount, B, G is two kinds of fluorescence (EGFP in blank group, DsRed) expression amount, experimental data adopts the process of SPSS statistical software, use one-way analysis of variance data, with Grap Pad Prism5 software, mapping analysis is carried out to normalization data.

Silencing efficiency to target gene when the present embodiment compares shRNA-Ago2 coexpression slow virus RNAi carrier and not same containing the shRNA single expression vector expression of Ago2 shRNA.With foreign gene DsRed for target gene, design 9 shRNA for its coding region, be connected to shRNA-Ago2 coexpression slow virus RNAi carrier involved in the present invention respectively, then be connected to respectively not containing the shRNA expression vector of Ago2.Detected the silencing efficiency of different shDsRed respectively by flow cytometer, result shows that the silence efficiency of 9 different shDsRed-Ago2 lentiviral vectorss is all apparently higher than the shDsRed expression vector (Fig. 2) not containing Ago2.

Embodiment 7shRNA-Ago2 coexpression slow virus RNAi carrier is to the silencing efficiency of native gene

The shRNA-Ago2 coexpression slow virus recombinant plasmid prepared for embodiment 5 carries out shRNA-Ago2 coexpression slow virus RNAi carrier to the experiment of the silencing efficiency of native gene, specifically comprises the following steps:

1.shRNA-Ago2 the slow virus packaging of coexpression slow virus RNAi carrier: with embodiment 6.

2. set up shRNA-Ago2 coexpression slow virus stable transfection Hela clone: with embodiment 6.

3.Western blot detects protein expression level:

(3.1) the Hela cell of cracking stable transfection goal gene pLVE-shRNA-Ago2 coexpression slow virus, collects protein sample, after carrying out quantification of protein, gets the protein example loading of 40 μ g, carry out SDS-PAGE electrophoresis in 100v constant voltage.

(3.2) transferring film: 200mA constant current 1.5h, after transferring film, is positioned over film in 5% skim-milk confining liquid and closes 1h, then with TBST, film is washed twice.

(3.3) add the ID1 primary antibodie (1:1000 dilution) with 3% skim-milk preparation, be positioned in shaking table, 4 DEG C are spent the night.

Within (3.4) second days, with TBST, film is washed three times, each 5min, add two anti-(the 1:5000 dilutions) of the mouse with 3% skim-milk preparation, carry out develop the color (or chemiluminescent autography reaction) after putting room temperature hybridization 1h.

The present embodiment designs 6 shRNA for the coding region of native gene ID1, after synthesis annealing, be connected respectively to pLVE-shRNA-Ago2 coexpression slow virus RNAi carrier involved in the present invention, be connected to respectively simultaneously not containing the shRNA expression vector of Ago2 as a comparison, then pack out corresponding shID1 slow virus liquid respectively; Virus liquid is infected Hela cell respectively, filters out Hela stably transfected cell line; Collect and lysing cell, by Western-Blot technology, protein ID1 is identified.Found that, in 6 kinds of shRNA, the silencing efficiency of the shRNA-Ago2 coexpression lentiviral vectors that the present invention relates to all obviously is better than not containing the shRNA single expression carrier (Fig. 3) of Ago2.

The structure of embodiment 8shRNA is on the impact of RNAi

Stem's length of shRNA, the factors such as the position of loop length and sense and antisense all can affect the efficiency of RNAi, in order to optimize shRNA project organization thus improve RNAi effect, the present embodiment designs respectively and constructs the shEGFP of the antisense-sense of stem's length is different, loop is different sense-antisense structure shEGFP and different loop.The oligonucleotide sequence of shRNA as shown in table 5 and table 6.

Table 5 is containing the shRNA oligonucleotide sequence of the target EGFP gene of different lengths

Table 6 is containing the shRNA oligonucleotide sequence of the target EGFP genes such as Different L oop length

Above-mentioned shEGFP result display after respectively transfected HEK 293, shRNA stem length 19 ~ 22nt all have more than 70% silencing efficiency (Fig. 4), wherein stem's length is that the silencing efficiency of 20nt is the strongest; Structure is that the shRNA of sense-antisense is stronger than the silencing efficiency of antisense-sense, and when in shRNA, loop length is 6 ~ 9nt, its interference effect is slightly better than 4nt(Fig. 5).Therefore, design shRNA time choose sense-antisen structure and by stem's cut to lengthen in the numerical control of 20nt, loop base built in 6 ~ 9nt, be so more conducive to the silencing efficiency of shRNA.

By optimum combination, the kind of the Expression element involved by shRNA-Ago2 coexpression slow virus RNAi carrier of the present invention, structure and array mode all have obvious enhancing for the silencing efficiency of shRNA.The shRNA-Ago2 coexpression slow virus RNAi carrier function that the present invention relates to is excellent and easy to use, high to the infection rate of higher eucaryotic cells, to the reticent effect lasts of goal gene, efficiently, facilitates investigator to apply RNAi technology better and conducts a research.

Embodiment 9 different promoters is on the impact of RNAi

For comparing the impact of different promoters on shRNA expression efficiency.The present embodiment constructs the shEGFP expression vector respectively containing promotor hU6, H1 and 7SK respectively, to observe its silencing efficiency to EGFP gene.The concrete construction process of shEGFP expression vector is substantially the same manner as Example 1, only lacks and inserts this step of enhancement factor Ago2 gene expression frame CMV-Ago2.By comparing the silencing efficiency of EGFP, find that hU6, H1 and 7SK promotor all can effectively drive shEGFP to express, interference effect is without significant difference (Fig. 6).

In addition, whether selectivity is existed to different promoters to analyze Ago2, the present embodiment also respectively with the pLVE-shRNA-Ago2 coexpression slow virus RNAi carrier that the present invention relates to for skeleton, build the shRNA-Ago2 coexpression slow virus RNAi carrier (i.e. embodiment 1-3 prepare shRNA-Ago2 coexpression slow virus RNAi carrier) respectively containing hU6, H1 and 7SK promotor, compare the selectivity of Ago2 to promotor further.Found that, for same promotor, pLVE-shRNA-Ago2 coexpression slow virus RNAi carrier is obviously than the silencing efficiency strong (Fig. 6) of single expression shEGFP carrier, illustrate that the silencing efficiency of Ago2 to the shRNA expression vector respectively containing these promotors all has enhancement, therefore there is not selectivity to different promoters in Ago2.Comprehensive above-mentioned experimental result, because Ago2 all has obvious enhancement to the silencing efficiency of the shRNA that hU6, H1 or 7SK drive, therefore the promotor driving shRNA to express in the shRNA-Ago2 coexpression slow virus RNAi carrier that builds of the present invention experimentally can need to choose hU6, H1 or 7SK one wherein and carries out vector construction.

Should be understood that, the invention is not restricted to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, all these improve and convert the protection domain that all should belong to claims of the present invention.

Claims (9)

1. a shRNA-Ago2 coexpression slow virus RNAi carrier, is characterized in that, described RNAi carrier comprises shRNA expression cassette and Ago2 protein expression frame; Described shRNA expression cassette comprises promotor hU6, H1 or 7SK, and ccdB lethal gene; Described Ago2 protein expression frame comprises CMV promoter, Ago2 protein-encoding gene, internal ribosome entry site DNA sequence dna and ZsGreen1 egfp expression gene.

2. shRNA-Ago2 coexpression slow virus RNAi carrier according to claim 1, it is characterized in that, the two ends of described ccdB lethal gene are provided with restriction enzyme site BamHI and MluI.

3. the construction process of the shRNA-Ago2 coexpression slow virus RNAi carrier described in any one of claim 1-2, is characterized in that, comprise the following steps:

(1) for the preparation of the skeleton pLVX-IRES-ZsGreen1-Puro of carrier construction:

With pLVX-IRES-ZsGreen1 carrier for template, amplification obtains IRES-ZsGreen1 fragment, by Cla1-Xba1 double digestion PCR primer, is then cloned into pLVX-Puro carrier by corresponding site, obtains pLVX-IRES-ZsGreen1-Puro carrier framework.

(2) enhancement factor Ago2 gene expression frame CMV-Ago2 is inserted:

With MluI and EcoRI double digestion pIRESneo-FLAG/HA-Ago2 carrier, obtain CMV-Ago2 fragment, the pLVX-IRES-ZsGreen1-Puro carrier framework that CMV-Ago2 fragment and step (1) reclaim is connected, proceed to competent escherichia coli cell, be coated with LB dull and stereotyped, overnight incubation, carries out bacterium colony PCR and enzyme cuts qualification, the carrier pLVX-CMV-Ago2-IRES-ZsGreen1-Puro obtained;

(3) insert shRNA and express promotor

With the carrier pLVX-CMV-Ago2-IRES-ZsGreen1-Puro that BclI and MluI double digestion step (2) obtains, dephosphorization acid rear electrophoresis reclaims; After PCR primer by the amplification of BclI and MluI double digestion hU6, H1 or 7SK promotor, be connected on the corresponding site of pLVX-CMV-Ago2-IRES-ZsGreen1-Puro carrier, proceed in competent escherichia coli cell, be coated with LB dull and stereotyped, overnight incubation, the carrier obtained after identifying is: pLVX-hU6/H1/7SK-CMV-Ago2-IRES-ZsGreen1-Puro.

(4) insert ccdB sequence and obtain shRNA-Ago2 coexpression slow virus RNAi carrier:

The fragment of intestinal bacteria ccdB lethal gene is contained from pLenti6 vector amplification, be connected with the pLVX-hU6/H1/7SK-CMV-Ago2-IRES-ZsGreen1-Puro carrier through BamHI and Mlu I double digestion with after BamHI with Mlu I double digestion, proceed in competent escherichia coli cell, be coated with the dual anti-flat board of LB/amp+Chl, overnight incubation, carry out bacterium colony PCR and enzyme cuts qualification, obtain shRNA-Ago2 coexpression slow virus RNAi carrier, carrier framework is pLVX-hU6/H1/7SK-ccdB-CMV-Ago2-IRES-ZsGreen1-Puro.

4. a shRNA-Ago2 coexpression slow virus recombinant plasmid, is characterized in that, comprises the shRNA-Ago2 coexpression slow virus RNAi carrier described in any one of claim 1-2.

5. shRNA-Ago2 coexpression slow virus recombinant plasmid according to claim 4, is characterized in that, also comprise goal gene shRNA, and stem's length of described shRNA is 19 ~ 22bp, and the loop length of described shRNA is 4 ~ 9bp.

6. shRNA-Ago2 coexpression slow virus recombinant plasmid according to claim 5, is characterized in that, stem's length of described shRNA is 20bp.

7. shRNA-Ago2 coexpression slow virus recombinant plasmid according to claim 5, it is characterized in that, the loop length of described shRNA is 6 ~ 9bp.

8. shRNA-Ago2 coexpression slow virus recombinant plasmid according to claim 5, is characterized in that, the sense/antisense structure of described shRNA is the sense-loop-antisense structure from 5 ' end.

9. the construction process of the shRNA-Ago2 coexpression slow virus recombinant plasmid described in claim 4 or 5, is characterized in that, comprise the following steps:

1) for goal gene design shRNA encoding sequence, two primer annealings are formed the shRNA of band restriction enzyme BamHI and MluI cohesive terminus by chemosynthesis two primers;

2) digest shRNA-Ago2 coexpression slow virus RNAi carrier skeleton by restriction enzyme BamHI and MluI, after enzyme cuts back to close, carrier framework is connected with the shRNA of annealing, transformation of E. coli competent cell coated plate overnight incubation;

3) the mono-clonal bacterium colony that flat board grows is carried out DNA sequencing, determine the correct insertion of shRNA encoding sequence, obtain shRNA-Ago2 coexpression slow virus recombinant plasmid.