CN106148408A - A kind of carrier system based on GFAP promoter and slow virus carrier and application thereof - Google Patents
A kind of carrier system based on GFAP promoter and slow virus carrier and application thereof Download PDFInfo
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Abstract
The invention belongs to genetic engineering and medical science.Specifically, the present invention relates to a kind of carrier system based on GFAP promoter and slow virus carrier, utilize GFAP promoter as the promoter in slow virus carrier.Present invention also offers this carrier system and obtain preparation method and application.Internal and vitro detection result shows, GFAP promoter can mediate the expression at HSCs for the luciferase, illustrating that the GFAP promoter of people can be as the expression of luciferase in slow virus delivery system special target positioning mouse HSC, the gene therapy for liver fibrosis provides potential clinical practice.
Description
Technical field
The invention belongs to biomedicine and field of gene, relate to a kind of liver star based on GFAP promoter and slow virus carrier
Shape cell-specific genes transduction method and application thereof.
Background technology
Liver fibrosis is the common trait of most of chronic liver disease, and main performance is a large amount of accumulation of extracellular matrix, and liver star
Shape cell is the main cell [1] participating in this process.HSCs is mainly distributed in sinus hepaticus gap, is close to hepatic sinusoidal endothelium
Cell, accounts for the 5%-10% of cell total amount in liver.In normal hepatocytes, HSCs is in quiescent condition, and rich in fat drip,
Vitamin A and retinol etc., when being caused, by virus, toxin, alcohol etc., the hepatic injury that liver fibrosis occurs, tranquillization
HSCs will change into mechanocyte, and in the process, HSCs generation form and function change,
Such as increase of propagation, shrinkage, chemotaxis, collagen and cell factor etc. [2], the deposition of too much collagen causes carefully
The substantial amounts of accumulation of extracellular matrix and liver fibrosis.
The activation of HSCs is the main process in liver fibrosis generating process, and therefore HSCs is that anti-fibrosis is controlled
The important target cell treated, main treatment means include the suppression of Hepatic Stellate Cell Activation, disappearing of the HSCs of activation
Remove, the degraded of extracellular matrix and the treatment of cell factor etc. [3-4].There are some researches show to promote liver fibrosis thin
The knocking out of HSCs of targeting of intracellular cytokine TGF β, PDGF and TNF-α can be as the method for Gene Therapy of Liver Cirrhosis
【5-7】.Such as, methods such as [9] is disturbed to reduce the table of TGF β by natural inhibitor [7], antibody [8] and RNA
Reach or suppress the activity of this signal path, the expression of collagen and the deposition of extracellular matrix can be reduced.
In the gene therapy of the HSCs targeting of liver fibrosis, the delivery system of cell-specific and high efficiency is very
Crucial, the medicament of some anti-fibrosis then can not be by the targeting positioning HSCs of cell-specific.Therefore, liver is starlike
The promoter of cell-specific starts the special gene of HSCs can capture difficulty.GFAP GFAP is one
Plant median fiber, specificity overexpression [10] in the astroglia of central nervous system.There are some researches show the GFAP of people
Promoter can be with the expression of the special lacZ reporter gene of gene transfer central nervous system of mice astroglia
【11】.Compared with other liver cells in liver, GFAP is also [12-13] of specificity overexpression in HSCs.With
During the DNA murine of herpes simplex virus thymidine kinase gene that at GCV, the GFAP promoter of reason mouse starts, can
So that the HSCs specific deficiency [14] of activation.In addition, transcript can be produced by what GFAP promoter mediated
(pri-miRNA) plasmid strikes and subtracts the expression of TGF-β in HSCs and can significantly reduce TGF-β 1 and collagen at egg
The expression [6] of white and rna level.And, the plasmid of the PDGF-B expression R-β shRNA being started by GFAP promoter leads to
The rotaring transfecting mode crossing hydrodynamics (HBT) can specifically strike in the acute liver injury of rats subtracting tetrachloro-methane induction
The expression [5] of PDGFR-β gene.Therefore, efficient, special, stable delivery system become this area study hotspot it
One.
The bibliography relevant with the present invention:
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Physiol.2013.3(4):1473-1492
2.Senoo H,Yoshikawa K,Morii M,Miura M,Imai K et al.Hepatic stellate cell(vitamin
A-storing cell)and its relative--past,present and future.Cell Biol
Int.2010.34(12):1247-1272
3.Hellemans K,Michalik L,Dittie A,Knorr A,Rombouts K et al.Peroxisome
proliferator-activated receptor-beta signaling contributes to enhanced
proliferation of hepatic stellate cells.Gastroenterology.2003.124(1):184-201.
4.Friedman SL,Sheppard D,Duffield JS,Violette S.Therapy for fibrotic diseases:
nearing the starting line.Sci Transl Med.2013.5(167):167sr161.
5.Chen SW,Chen YX,Zhang XR,Qian H,Chen WZ et al.Targeted inhibition of
platelet-derived growth factor receptor-beta subunit in hepatic stellate cells
ameliorates hepatic fibrosis in rats.Gene Ther.2008.15(21):1424-1435.
6.Yang N,Mahato RI.GFAP promoter-driven RNA interference on TGF-beta1 to treat
liver fibrosis.Pharm Res.2011.28(4):752-761.
7.Baghy K,Iozzo RV,Kovalszky I.Decorin-TGFbeta axis in hepatic fibrosis and
cirrhosis.J Histochem Cytochem.2012.60(4):262-268.
8.Zhang DW,Zhao YX,Wei D,Li YL,Zhang Y et al.HAb18G/CD147promotes activation
of hepatic stellate cells and is a target for antibody therapy of liver fibrosis.
J Hepatol.2012.57(6):1283-1291.
9.Ogawa T,Iizuka M,Sekiya Y,Yoshizato K,Ikeda K et al.Suppression of type
I collagen production by microRNA-29b in cultured human stellate cells.Biochem
Biophys Res Commun.2010.391(1):316-321.
10.Bignami A,Dahl D.The astroglial response to stabbing.Immunofluorescence
studies with antibodies to astrocyte-specific protein(GFA)in mammalian and
submammalian vertebrates.Neuropathology and Applied Neurobiology.1976.2(2):
99-110.
11.Brenner M,Kisseberth WC,Su Y,Besnard F,Messing A.GFAP promoter directs
astrocyte-specific expression in transgenic mice.J Neurosci.1994.14(3 Pt 1):
1030-1037.
12.Maubach G,Lim MC,Zhang CY,Zhuo L.GFAP promoter directs lacZ expression
specifically in a rat hepatic stellate cell line.World J Gastroenterol.2006.12(5):
723-730.
13.Russo FP,Alison MR,Bigger BW,Amofah E,Florou A et al.The bone marrow
functionally contributes to liver fibrosis.Gastroenterology.2006.130(6):
1807-1821.
14.Puche JE,Lee YA,Jiao J,Aloman C,Fiel MI et al.A novel murine model to deplete
hepatic stellate cells uncovers their role in amplifying liver damage in mice.
Hepatology.2013.57(1):339-350.
15.Wolff JA,Budker V.The mechanism of naked DNA uptake and expression.Adv
Genet.2005.54:3-20.
16.Zhang G,Gao X,Song YK,Vollmer R,Stolz DB et al.Hydroporation as the mechanism
of hydrodynamic delivery.Gene Ther.2004.11(8):675-682.
17.Azzam T,Domb AJ.Current developments in gene transfection agents.Curr Drug
Deliv.2004.1(2):165-193.
18.Delzor A,Escartin C,Deglon N.Lentiviral vectors:a powerful tool to target
astrocytes in vivo.Curr Drug Targets.2013.14(11):1336-1346.
19.Guo CJ,Pan Q,Jiang B,Chen GY,Li DG.Effects of upregulated expression of
microRNA-16 on biological properties of culture-activated hepatic stellate cells.
Apoptosis.2009.14(11):1331-1340.
20.Clement S,Pascarella S,Conzelmann S,Gonelle-Gispert C,Guilloux K et al.The
hepatitis C virus core protein indirectly induces alpha-smooth muscle actin
expression in hepatic stellate cells via interleukin-8.J Hepatol.2010.52(5):
635-643.
21.Xu L,Hui AY,Albanis E,Arthur MJ,O'Byrne SM et al.Human hepatic stellate
cell lines,LX-1 and LX-2:new tools for analysis of hepatic fibrosis.
Gut.2005.54(1):142-151.
22.Guo J,Loke J,Zheng F,Hong F,Yea S et al.Functional linkage of
cirrhosis-predictive single nucleotide polymorphisms of Toll-like receptor 4 to
hepatic stellate cell responses.Hepatology.2009.49(3):960-968.
23.Hernandez-Gea V,Ghiassi-Nejad Z,Rozenfeld R,Gordon R,Fiel MI et al.Autophagy
releases lipid that promotes fibrogenesis by activated hepatic stellate cells in
mice and in human tissues.Gastroenterology.2012.142(4):938-946.。
Content of the invention
It is an object of the invention to provide a kind of carrier system based on GFAP promoter and slow virus carrier.
It is a further object to provide preparation and the application of above-mentioned carrier system.
The gene delivery mode of non-viral transfection efficiency in mouse and rat higher [15] [5], but HBT mode is in reality
Test and all have limitation in clinical practice.Gymnoplasm grain in host cell can only transient expression, and in order to maintain internal external source base
The stable expression of cause is accomplished by constantly repeating to give, and will increase vein pressure, liver through the quickly injection in a large number of tail vein in HBT
Infarct, of short duration cardiac shock is even dead [16].Virus gene delivery vehicle such as slow virus carrier then can have very high passing
Sending efficiency and continual and steady gene expression, the damage to animal bodies is relatively low, but also has potential biohazard, can
Can increase immunogenicity [17-18].Lentiviral can successfully be delivered to brain astrocytes and
Expression alien gene [18-20] in HSCs in liver.The integration of cell specificity promotor can be at astroglia
Middle selective expression's foreign gene [18].But the specific efficient table of the internal foreign gene being mediated by GFAP promoter
The lentiviral gene delivery system reaching also did not detected in HSCs, and therefore the present invention constructs by GFAP
The slow virus carrier of promoter mediation, utilizes luciferase as Testing index, and is delivered to mouse by tail vein injection
In liver, the then activity of luciferase and expression in detection body, after result is shown in tail vein injection, the GFAP promoter of people
Can mediate the specific expressed of luciferase in Mouse Liver sternzellen, prompting GFAP promoter and slow virus deliver body
Gene therapy for liver fibrosis is provided potential clinical practice by the integration of system.The present invention completes on this basis.
The invention provides a kind of carrier system based on GFAP promoter and slow virus carrier, utilize GFAP promoter conduct
Promoter in slow virus carrier.
In one embodiment of the invention, primer sequence such as SEQ ID the NO 1 and SEQ ID NO of GFAP promoter is expanded
Shown in 2.
Described GFAP promoter is people's GFAP promoter or mouse GFAP promoter.
The invention provides the preparation method of above-mentioned carrier system, i.e. utilize GFAP promoter as the startup in slow virus carrier
Son.The method comprises the following steps:
(1) expand and obtain the DNA fragmentation of GFAP promoter；
(2) it is cloned into the DNA fragmentation of GFAP promoter in slow virus carrier.
Wherein, in step (1), the amplimer of GFAP promoter can use such as SEQ ID NO 1 and SEQ ID NO 2
Shown primer.
Step in (2) is cloned into GFAP promoter in pGL3-Basic or pCDH-GFP carrier.PGL3-Basic or
Person's pCDH-GFP carrier all can be by commercially available acquisition.
Preferably, ATG in described GFAP promoter can be sported TTG.
On the other hand, the invention provides the application of described carrier system, i.e. utilize this carrier system to import foreign gene
The cell of in vitro culture.Preferably, described cell is HSCs.
In one embodiment of the invention, construct the slow virus carrier being mediated by GFAP promoter, utilize fluorescein
Enzyme, as Testing index, detects the spy of the transduction to foreign gene for the slow virus carrier being mediated by GFAP promoter and expression
The opposite sex and validity.Result all detects the expression of specificity fluorescent element enzyme in HSCs and Mouse Liver.
The present invention constructs the slow virus carrier system comprising GFAP promoter, and have detected this system in vivo and in vitro and exist
The gene transfer of HSCs and the specific and validity of expression.It is little that we also demonstrate through tail vein injection slow virus
The expression of the HSCs specificity fluorescent element enzyme of GFAP promoter mediation in mouse liver, we are result display GFAP
The slow virus carrier of promoter mediation is highly useful instrument in genetic manipulation and the gene therapy for liver fibrosis carries
For potential clinical practice.
Brief description
Fig. 1: the mediation of the GFAP promoter of people is in hgher efficiency in LX-2 and JS1 cell.
PGL3-basic, pGL3-hGFAP-p-luci, pGL3-mGFAP-p-luci and pGL3-promoter are transfected
After LX-2 and JS1 cell, with the expression of double reporter gene detection luciferase, wherein sea cucumber luciferase makees
For internal reference, pGL3-basic is as negative control, and pGL3-promoter is as positive control.All data analyses are all roots
Independently repeat experiment according to three times and complete (* * P < 0.01, * P < 0.05).
Fig. 2: three kinds of virus infectious effect to L-02, JS1 and LX-2 cell.
All pictures are all to shoot under fluorescence microscope, and green fluorescence picture is presented herein below its white light picture, picture multiplication factor
Be 200 ×.
Fig. 3: expression and the activity of the luciferase of the GFAP promoter mediation of people shows in JS1 and LX-2 cell
Write and be higher than L-02 cell.The expression of luciferase in A.Western blot checking L-02, JS1 and LX-2 cell.B.
It is to calculate gray value with software I mage J, the relative value expressed as the luciferase that internal reference obtains using β-actin.
C. be the mediated effects by single reporter gene detection GFAP promoter, using the CMV promoter of same cell as
Internal reference, the activity of the luciferase being mediated by GFAP promoter in JS1 and LX-2 is significantly higher than L-02 cell
(* * P < 0.01, * P < 0.05).
Fig. 4: the distribution map of virus in Mouse Liver.
By having injected 3 kinds of viral sacrifice, take liver and do freezing microtome section, by double fluorescent stainings detection luciferase and
The expression of GFAP.Fluorescence blue in a, b, c, d represents GFAP albumen；In e, f, g, h, red fluorescence represents luciferase；
I, j, k, l be merge after picture (a and e merge obtain i, b and f merge obtain j, c and g merge obtain k, d and h
Merge and obtain l)；M, n, o, p Green fluorescence represents GFP；In q, r, s, t, purple light represents DAPI, is shown that nucleus institute
In position.Liver for the mouse having injected pCDH-hGFAP-p-luci virus has been cooked portal area (the 3rd row) and pars affecta
The detection of position (the 4th row).
The expression figure of luciferase in Fig. 5: GFAP promoter targeting HSCs
Double immunofluorescence experiment detection luciferase is done to the liver slice of the mouse having injected pCDH-GFAP-p-luci virus
With Desmin (A) and α-Sma (B).A. yellow arrows represents portal area, and white arrow represents damage field.
Detailed description of the invention
In the gene therapy of liver fibrosis, HSCs (HSCs) is a kind of very important target cell, and liver is starlike carefully
Born of the same parents' special glial fibrillary acidic protein (GFAP) promoter can be in transgenic mice outside mediated targeted HSCs
The expression of source gene.In our current research, we construct the slow virus carrier of the luciferase comprising to be mediated by GFAP promoter,
And have detected the specificity and efficiency of the exogenous gene expression of inside and outside HSCs targeting.In vitro, we select Jie
Lead the GFAP promoter of the higher people of efficiency, and mediate luciferase with CMV promoter respectively as positive control and exist
Expression in Mouse Liver sternzellen (JS1), human liver microsome proteins (LX-2) and human liver cell (L02).In vivo,
The viral vectors of packaging is expelled in the liver fibrosis Mice Body of tetrachloro-methane induction by tail vein injection by we, by work
Body imaging and WB detection distribution situation in Mice Body for the slow virus carrier, and by immunity positioning altogether, as HSCs is special
The special expression of opposite sex marker detection HSCs.Result shows, GFAP promoter can mediate fluorescein
Enzyme is in the expression of HSCs, and in vivo, virus is mainly distributed on mouse web portion, and the GFAP promoter of people is described
Can be that the gene of liver fibrosis is controlled as the expression of luciferase in slow virus delivery system special target positioning mouse HSC
Treat and potential clinical practice is provided.
Below in conjunction with preferred embodiment, the present invention is elaborated, without limiting the present invention.
Material and method:
Clone:
Human liver microsome proteins system LX-2[21], Mouse Liver sternzellen JS1[22-23], human liver cell L02 and human embryo kidney
Cell HEK-293T.All cells is all cultivated by the DMEM of 10%FBS.
The structure of the reporter plasmid that embodiment 1 is mediated by GFAP promoter
Construct the reporter plasmid being mediated by GFAP promoter and slow virus plasmid according to previous research [11].From people
Expanding the fragment of GFAP promoter on genome, primer is F:5 '-tcactgcttacgcccaggtc-3 ' (SEQ ID
NO 1), R:5 '-gcgagcagcggaggtgat3 ' (SEQ ID NO 2), after reclaiming through rubber tapping, with p-GEM-T Easy
Vector connects, after Plastid transformation, the screening of blue hickie, choose bacterium and shake after bacterium order-checking identifies, extract plasmid.With
PEGM-T-HGFAP-promoter is template, expands Hgfap-p sequence, and primer is: h-GFAP-P-NheI-F:
5 '-cctcgctagc (NheI) tcactgcttacgcccaggtc-3 ' (SEQ ID NO 3), h-GFAP-P-HindIII-R:
5 '-cctcaagctt (HindIII) gcgagcagcggaggtgat (SEQ ID NO 4)-3 ', after reclaiming through rubber tapping,
With NheI and HindII I restriction enzymes double zyme cutting, with NheI and HindIII restriction enzymes double zyme cutting
PGL3-Basic connects, through Plastid transformation, coated plate, choose bacterium, shake bacterium, bacterium colony PCR identifies, order-checking qualification etc. obtains plasmid
pGL3-NheI-hGFAP-promoter-HindIII-Basic.Which is template, as follows with primer: F:
5 '-agcagagccagagcaggttggagaggagacgcatca-3 ' (SEQ ID NO 5), R:
5 '-tgatgcgtctcctctccaacctgctctggctctgct-3 ' (SEQ ID NO 6), after being digested with DpnI, turn
Change, coated plate, choose bacterium, shake bacterium, order-checking identify, obtain plasmid
PGL3-NheI-mutant-hGFAP-promoter-HindIII-Basic, i.e. pGL3-hGFAP-P-luci.GFAP
In promoter district, ATG sports TTG.Same method is through amplimer F:5 '-tctccagcgttgctgacaaacct-3 '
(SEQ ID NO 7), R:5 '-gcggcgcgcagaggtgatg-3 ' (SEQ ID NO 8) synthesizes
mouse-GFAP-promoter；Through being digested primer M-GFAP-P-NheI-F:
5 '-cctcgctagctctccagcgttgctgacaaacct-3 ' (SEQ ID NO 9): M-GFAP-P-HindIII-R:
5 '-cctcaagcttgcggcgcgcagaggtgatg-3 ' (SEQ ID NO 10), obtain plasmid
PGL3-NheI-mouseGFAP-promoter-HindIII-Basic, then through point mutation primers F:
5 '-gatgcgtctccgctccaacctgccctgcctctgct-3 ' (SEQ ID NO 11), R:
5 '-agcagaggcagggcaggttggagcggagacgcatc-3 ' (SEQ ID NO 12) obtain
PGL3-NheI-mutant-mGFAP-promoter-HindIII-Basic, i.e. pGL3-mGFAP-P-luci.
With plasmid pGL3-NheI-mutant-hGFAP-promoter-HindIII-Basic as template, with primers F:
5 '-cctcactagttcactgcttacgcccaggtc-3 ' (SEQ ID NO 13), R:
5 '-ggagctgactgggttgaag-3 ' (SEQ ID NO 14), amplification obtains the DNA piece containing SpeI restriction enzyme site
Section, after reclaiming through rubber tapping, with SpeI and BamHI restriction enzymes double zyme cutting, restricted interior with SpeI and BamHI
Cut enzyme double digestion pCDH-GFP connect, through Plastid transformation, coated plate, choose bacterium, shake bacterium, bacterium colony PCR identify, order-checking identify
Etc. obtaining plasmid pCDH-hGFAP-P-luci, substituted for the CMVpromoter of pCDH-GFP, and by the piece of luciferase
Section is inserted in carrier.Build plasmid positive control group be with NheI and BamHI restriction enzyme respectively by pGL3-Basic,
After pCDH-GFP double digestion, reclaim through PCR cleaning, connections, conversion, coated plate, choose bacterium, shake bacterium, the qualification etc. of checking order obtains
Plasmid pCDH-CMV-luci, does not change the promoter of carrier itself.Negative control group is plasmid pCDH-GFP.
Embodiment 2 Reporter Gene Experiments
LX-2 and JS1 cell is according to every hole 105Individual cell carries out 24 orifice plate bed boards, after cultivating 24 hours, transfects respectively
PGL3-hGFAP-p-luci, pGL3-mGFAP-p-luci and negative control pGL3-basic and positive control
PGL3-promoter (luciferase driven by SV-40promoter), every hole 0.8ug, be used in combination
Lipofectamine2000 cotransfection internal reference pRL-CMV (luciferase driven by CMV promoter), every hole 8ng,
Each all does 4 multiple holes, repeats for 3 times.Give a report after 24-48 hour Gene Experiments, first washed twice by 1*PBS, blot
Liquid, with 1*passive lysis buffer lysis at room temperature 15 minutes, then extracts the double reporter gene detection bodies of supernatant
The activity of system's detection luciferase.
Embodiment 3 Infection in Vitro
By virus packaging plasmid psPAX2 and pSD respectively with plasmid pCDH-hGFAP-p-Luci, pCDH-CMV-Luci and
PCDH-GFP cotransfection 293T cell, collects supernatant, and 2000g centrifuges 5 minutes, and filters with 0.45um filter, takes
A part of virus infects JS1, LX-2 and L02 respectively, and another part then through 25000g ultracentrifugation 2 hours, obtains
White precipitate PBS is frozen at-80 DEG C after dissolving, for virus titer detection and tail vein injection.Examine under a microscope
After the cell of 90% shows green glow under fluorescence microscope, single reporter gene detection can be done, and collect Protein Detection luciferase
Express.
As also illustrated in figs. 2 a-b, although the mediated effects of GFAP promoter does not has CMV promoter strong, but JS1 and
The expression of the luciferase of GFAP promoter mediation in two kinds of cells of LX-2 is significantly higher than the expression in L02 cell.Single
Reporter gene result as shown in Figure 2 C, the work of the luciferase being mediated by GFAP promoter in two kinds of cells of JS1 and LX-2
Property be also significantly greater than L02 cell, this shows to make the slow virus delivery system of the GFAP promoter of employment can be special in vitro
The expression of gene in opposite sex targeting mediation HSCs.
The foundation of Liver Fibrosis Model and infection in embodiment 4 body
Taking 4 weeks big BalB/C male mices, body weight is approximately 20g, is randomly divided into two groups, one group of mouse peritoneal injection olive oil
As negative control, another group mouse according to the volume ratio lumbar injection of carbon tetrachloride and olive oil 1:4,5ul/ug for the first time,
Remaining is all 2.5ul/ug, and injection in every 3 days is once.After 4 weeks, randomly select 2 inducing mouses, take liver and do Picro-Sirius red
Dyeing carries out the detection of Level of Hepatic Fibrosis.Then two groups of mouse are respectively randomly divided into 3 groups, and inject respectively
LV-pCDH-hGFAP-p-Luci, LV-pCDH-CMV-Luci and LV-pCDH-GFP, dosage is every mouse 2*107Tu, often
It is once injected twice.All mouse are provided by Medical Center of Fudan University's Experimental Animal Center, and all experiments all meet multiple
The requirement of denier University Medical College animal used as test ethics association.
Living imaging in embodiment 5 body
After injecting virus 12 days, lumbar injection D-luciferin, uses IVIS imaging after dosage 150mg/kg, 10-15 minute
In system detection Mice Body, luciferase is substantially distributed in abdominal cavity, then puts to death mouse and takes liver, kidney, and detection GFP expresses.
The Protein Detection of embodiment 6 luciferase and green fluorescent protein and Immunofluorescence test
It is embedded in the hepatic tissue of acquirement in OCT and is quickly put in cryocoagulation in liquid nitrogen, then frozen do freezing microtome section in-80 DEG C,
Experimental implementation process guarantees that the sampling point of every mouse is as far as possible identical.Freezing microtome section is put in the paraformaldehyde of 4% and fixes 15
Minute, wash 3 times with TBS-T, each 3-5 minute, closed 1 hour by immunofluorescence confining liquid, then resist with one and incubate
Educate 4 DEG C overnight, through TBS-T wash 3 times, each 3-5 minute, add the anti-incubated at room of fluorescence two 1 hour, then use TBS-T
Wash 3 times, each 3-5 minute, finally taken pictures by fluorescence microscope.Because of mouse respectively organize between there is no suitable internal reference, first
After BCA kit, then show Tot Prot with Coomassie brilliant blue, finally do the content of GFP in WB detection liver kidney,
Expression with display virus position.
SEQUENCE LISTING
<110>Fudan University
<120>a kind of carrier system based on GFAP promoter and slow virus carrier
<130> 20150308
<160> 14
<170> PatentIn version
3.3
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<211> 20
<212> DNA
<213> Artificial
<220>
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<400> 1
tcactgctta cgcccaggtc 20
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gcgagcagcg gaggtgat
18
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<223> primer
<400> 3
cctcgctagc tcactgctta cgcccaggtc 30
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<211> 28
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<213> Artificial
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cctcaagctt gcgagcagcg gaggtgat 28
<210> 5
<211> 36
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<213> Artificial
<220>
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<400> 5
agcagagcca gagcaggttg gagaggagac gcatca 36
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tgatgcgtct cctctccaac ctgctctggc tctgct 36
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<211> 23
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<213> Artificial
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<223> primer
<400> 7
tctccagcgt tgctgacaaa cct 23
<210> 8
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<213> Artificial
<220>
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<400> 8
gcggcgcgca gaggtgatg
19
<210> 9
<211> 33
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<213> Artificial
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<400> 9
cctcgctagc tctccagcgt tgctgacaaa cct 33
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agcagaggca gggcaggttg gagcggagac gcatc 35
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<400> 13
cctcactagt tcactgctta cgcccaggtc 30
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<400> 14
ggagctgact gggttgaag
19
Claims (10)
1. the carrier system based on GFAP promoter and slow virus carrier, it is characterised in that utilize GFAP promoter to make
For the promoter in slow virus carrier.
2. carrier system as claimed in claim 1, it is characterised in that the primer sequence such as SEQ ID of amplification GFAP promoter
Shown in NO 1 and SEQ ID NO 2.
3. carrier system as claimed in claim 1, it is characterised in that described GFAP promoter is people's GFAP promoter
Or mouse GFAP promoter.
4. the preparation method of carrier system as claimed in claim 1, it is characterised in that utilize GFAP promoter as slowly
Promoter in viral vectors.
5. preparation method as claimed in claim 4, it is characterised in that the method comprises the following steps:
(1) expand and obtain the DNA fragmentation of GFAP promoter；
(2) it is cloned into the DNA fragmentation of GFAP promoter in slow virus carrier.
6. preparation method as claimed in claim 5, it is characterised in that the amplimer of GFAP promoter in step (1)
As shown in SEQ ID NO 1 and SEQ ID NO 2.
7. preparation method as claimed in claim 5, it is characterised in that GFAP promoter is cloned in (2) by step
In pGL3-Basic or pCDH-GFP carrier.
8. preparation method as claimed in claim 4, it is characterised in that in described GFAP promoter, ATG sports TTG.
9. the application of carrier system as claimed in claim 1, it is characterised in that utilize this carrier system to lead foreign gene
Enter the cell of in vitro culture.
10. apply as claimed in claim 9, it is characterised in that described cell is HSCs.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660154A (en) * | 2017-03-29 | 2018-10-16 | 深圳市人民医院 | A kind of carrier system of high specifically expressing target gene efficient in liver cancer cells |
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| CN1759182A (en) * | 2002-11-22 | 2006-04-12 | 克雷顿研究院 | Compositions and systems for the regulation of genes |
| CN103305514A (en) * | 2013-06-24 | 2013-09-18 | 南京医科大学 | Mouse astrocyte specific promoter and lentiviral vector thereof |
| CN103667190A (en) * | 2012-09-07 | 2014-03-26 | 上海吉凯基因化学技术有限公司 | Method for forming nerve cells by induction and composition |
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| CN1759182A (en) * | 2002-11-22 | 2006-04-12 | 克雷顿研究院 | Compositions and systems for the regulation of genes |
| CN103667190A (en) * | 2012-09-07 | 2014-03-26 | 上海吉凯基因化学技术有限公司 | Method for forming nerve cells by induction and composition |
| CN103305514A (en) * | 2013-06-24 | 2013-09-18 | 南京医科大学 | Mouse astrocyte specific promoter and lentiviral vector thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108660154A (en) * | 2017-03-29 | 2018-10-16 | 深圳市人民医院 | A kind of carrier system of high specifically expressing target gene efficient in liver cancer cells |
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