CN104059943A - Panx3 gene overexpression red fluorescence lentiviral vector and building method and application thereof - Google Patents
Panx3 gene overexpression red fluorescence lentiviral vector and building method and application thereof Download PDFInfo
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Abstract
The invention discloses a Panx3 gene overexpression red fluorescence lentiviral vector and a building method and application of the Panx3 gene overexpression red fluorescence lentiviral vector, and belongs to the field of gene engineering. According to the vector, a lentiviral vector pLL3.7 is used as a skeleton vector, a Panx3 gene, CMV promoter and RFP gene segment is inserted behind a CMV promoter of pLL3.7 in the direction from the 5' end to the 3' end, and the sequence of the segment is shown in SEQIDNO.1. The built Panx3 gene overexpression red fluorescence lentiviral vector has the advantages that the mutual interference problem of the overexpression vector and function research in Panx3 gene research is solved, a CMV serves as the promoter of the vector, eukaryon gene efficient expression can be guaranteed, RFP serves as a marker, the role of the Panx3 gene in intracellular calcium and ATP change after a half-channel is formed can be directly observed, the cell function is eventually affected, and a good tool is provided for research on the functions of the Panx3 gene.
Description
Technical field
The invention belongs to genetically engineered field, relate to the structure of Panx3 gene overexpression lentiviral vectors, be specifically related to a kind of red fluorescence lentiviral vectors and construction process and application of Panx3 gene overexpression.
Background technology
Panx3 is that Panchin in 2000 etc. find a class inserted by connexin in vertebrates body, is one of the topmost inserted by connexin of vertebrates family.It can form gap albumen passage, allows ion and small-molecule substance to pass through, and then flanking cell and cell and extracellular matrix are interconnected, and is the movable synchronized basis of iuntercellular.At present, be mainly manifested in formation hemichannel for the function of Panx3 gene, transmit the signaling molecules such as calcium ion, cAMP and ATP, and then affect the functions such as downstream series of cell proliferation, differentiation.
Slow virus is to the common instrument plasmid of gene functional research in a kind of molecular biology, energy high-efficiency transfection protokaryon and eukaryotic cell, make cell can synthesize and express goal gene, by contrast goal gene at cell inner expression whether or expression intensity, realize to goal gene cell even the effect in body function verify and analyze.Therefore, slow virus is very extensive in the application of research gene in cell and body function.Because the function of Panx3 gene is main relevant to calcium ion, ATP etc., and the current application that all relates to green fluorescence for the research method of calcium ion and ATP, and the carrier of current correlative study design is non-fluorescence or green fluorescence sign, or can not directly observe transfection effect, or disturb mutually with the research that intracellular calcium and ATP change.Therefore distinguishing slow-virus transfection cell and intracellular calcium and ATP how intuitively, effectively changes and seems very important.
Summary of the invention
The object of the invention is to solve over-express vector and the mutual interference problem of functional study in Panx3 gene studies, a kind of red fluorescence lentiviral vectors of Panx3 gene overexpression is provided.
Another object of the present invention is to provide the construction process of above-mentioned carrier.
A further object of the present invention is to provide the application of above-mentioned carrier.
Object of the present invention is achieved through the following technical solutions:
A kind of red fluorescence lentiviral vectors of Panx3 gene overexpression, taking lentiviral vectors pLL3.7 as skeleton carrier, after the CMV of pLL3.7 promotor, insert Panx3 gene, CMV promotor and RFP(red fluorescent protein according to 5 ' to 3 ' direction) gene fragment (Panx3-CMV-RFP gene fragment).
Preferably, the sequence of described Panx3 gene, CMV promotor and RFP gene fragment is as shown in SEQ ID NO.1.
The construction process of the red fluorescence lentiviral vectors of above-mentioned Panx3 gene overexpression, comprise the steps: to design 5 ' and hold the primer that contains restriction enzyme digestion sites, increase respectively Panx3 gene, CMV promotor and RFP gene fragment, be connected to Panx3 gene, CMV promotor and RFP red fluorescent protein fragment on pLL3.7 according to restriction enzyme site and the lentiviral vectors pLL3.7 restriction enzyme site of institute's amplified fragments 5 ' end.
Preferably, the construction process of the red fluorescence lentiviral vectors of described Panx3 gene overexpression, comprises the steps:
(1) extract total RNA of people pulp cells, taking the cDNA of reverse transcription as template, pcr amplification Panx3 gene fragment, the primer is:
Primer 1:5 '-CCCAAGCTTATGTCACTTGCACACACAG-3 ',
Primer 2: 5 '-CGCAAGCTTTTTGACTCTTCGGCTCCA-3 ';
By HindIII restriction enzyme site, Panx3 gene is linked and on pCDNA3.1 (+), obtained pCDNA3.1 (+)-Panx3 recombinant plasmid.
(2) with shipping agency of Thy-Brainbrow-1.0H(Addgene China, Plasmid 18724) plasmid is template, pcr amplification RFP red fluorescent protein fragment, the primer is:
Primer 3:5 '-TCCCCCGGGAGACCCAAGCTGGCTAGCG-3 ',
Primer 4:5 '-CCCTTCGAACTGGCAACTAGAAGGCACAGTCG-3 ';
By SmaI and BstBI restriction enzyme site, RFP gene is linked and on pCDNA3.1 (+)-Panx3, obtained pCDNA3.1 (+)-Panx3-RFP recombinant plasmid.
(3) taking pCDNA3.1 (+) as template, pcr amplification CMV promoter fragment, the primer is:
Primer 5:5 '-CGCGGATCCGCTTGACCGACAATTGCAT-3 ',
Primer 6:5 '-CGCGGATCCATTTCGATAAGCCAGTAAGCAG-3 ';
By BamHI restriction enzyme site, CMV gene is linked and on pCDNA3.1 (+)-Panx3-RFP, obtained pCDNA3.1 (+)-Panx3-CMV-RFP recombinant plasmid.
(4) the Panx3-CMV-RFP fragment on pCDNA3.1 (+)-Panx3-CMV-RFP is linked to the red fluorescence lentiviral vectors (pLL3.7-CMV-Panx3-CMV-RFP) that obtains Panx3 gene overexpression on pLL3.7 by NheI and PmiI restriction enzyme site.
The application of the red fluorescence lentiviral vectors of above-mentioned Panx3 gene overexpression in research Panx3 gene function.
The present invention has following advantage and effect with respect to prior art:
The present invention has built a kind of red fluorescence lentiviral vectors of Panx3 gene overexpression, has solved over-express vector and the mutual interference problem of functional study in Panx3 gene studies.This lentiviral vectors is taking CMV as promotor, can ensure the high efficient expression of eukaryotic gene, employing RFP is marker, can intuitively observe Panx3 gene and form after hemichannel the effect in intracellular calcium and ATP change, the final cell function that affects, for the function of research Panx3 gene provides good instrument.
Brief description of the drawings
Fig. 1 is that pCDNA3.1 (+)-Panx3 recombinant plasmid NcoI and PvuII enzyme are cut qualification figure, and 1-8 is that 8 mono-clonal plasmid NcoI enzymes are cut, and 9-16 is that 8 mono-clonal plasmid PvuII enzymes are cut.
Fig. 2 is that pCDNA3.1 (+)-Panx3-RFP recombinant plasmid NcoI-HF enzyme is cut qualification figure, and 1-5 is that 5 mono-clonal plasmid NcoI-HF enzymes are cut, and M is 1kb Marker(Fermentas).
Fig. 3 is that pCDNA3.1 (+)-Panx3-CMV-RFP recombinant plasmid NcoI-HF enzyme is cut qualification figure, and 1-4 is that 4 mono-clonal plasmid NcoI-HF enzymes are cut, and M is 1kb Marker(Fermentas).
Fig. 4 is that pLL3.7-CMV-Panx3-CMV-RFP recombinant plasmid HindIII and NcoI enzyme are cut qualification figure, and 1-4 is that 4 mono-clonal plasmid HindIII enzymes are cut, and 5-8 is that 4 mono-clonal plasmid NcoI enzymes are cut, and M is 1kb Marker(Fermentas).
Fig. 5 is the red fluorescence lentiviral vectors pLL3.7-CMV-Panx3-CMV-RFP schematic diagram of Panx3 gene overexpression.
Fig. 6 is the fluorescence microscope pLL3.7-CMV-Panx3-CMV-RFP virus infection odontoblast visual field of 48 hours.
Fig. 7 is the result figure that real-time fluorescence quantitative PCR and Western blot analyze Panx3 mRNA and protein expression content after pLL3.7-CMV-Panx3-CMV-RFP virus infection odontoblast, A: real-time fluorescence quantitative PCR, B:Western blot.
Fig. 8 is the impact that fluorescence microscope cold stimulation discharges ATP in odontoblast, A: control group, B:Panx3 crosses expression group.
Fig. 9 is the histogram that after cold stimulation, in odontoblast, ATP discharges.
Embodiment
Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.Enzyme used, without special instruction, is NEB company product.
The structure of embodiment 1 pCDNA3.1 (+)-Panx3
From people's pulp cells, extract total RNA, taking the cDNA of reverse transcription as template, according to the primers of Panx3 gene cDNA, carry out pcr amplification taking primer 1 and primer 2 as primer,
Primer 1:5 '-CCC
aAGCTTaTGTCACTTGCACACACAG-3 ',
Primer 2: 5 '-CGC
aAGCTTtTTGACTCTTCGGCTCCA-3 ' (base with underscore is HindIII recognition site).
PCR reaction conditions is: 94 DEG C of 3min, 94 DEG C of 30s, 58 DEG C of 40s, 72 DEG C of 1min totally 35 circulations afterwards, last 72 DEG C of 10min.
PCR can obtain a linear DNA amplified production Panx3(1396bp), two ends are respectively with HindIII restriction enzyme site, with HindIII simultaneously enzyme cut Panx3 fragment and pCDNA3.1 (+) plasmid, gel reclaims test kit and reclaims (health is ShiJi Co., Ltd), T4 DNA ligase carries out two fragment connections, Transformed E .coli DH5 α competent cell (health is ShiJi Co., Ltd).Test kit extracting pCDNA3.1 (+)-Panx3 recombinant plasmid, restriction enzyme NcoI and PvuII enzyme are cut, and enzyme is cut product by 1.0% agarose gel electrophoresis qualification, electrophoresis result and in theory collection of illustrative plates identical (Fig. 1).Plasmid is delivered Shanghai Sheng Gong company and is checked order, and Panx3 gene sequencing result is correct.
The structure of embodiment 2 pCDNA3.1 (+)-Panx3-RFP
With shipping agency of Thy-Brainbrow-1.0H(Addgene China, Plasmid 18724) carrier is template, taking primer 3 and primer 4 for the primer PCR RFP red fluorescent protein fragment that increases.
Primer 3:5 '-TCC
cCCGGGaGACCCAAGCTGGCTAGCG-3 ', SmaI is identified as a little,
Primer 4:5 '-CCC
tTCGAAcTGGCAACTAGAAGGCACAGTCG-3 ', BstBI recognition site.
PCR reaction conditions is: 94 DEG C of 3min, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min totally 33 circulations afterwards, last 72 DEG C of 10min.
PCR obtains a linear DNA amplified production RFP(805bp), one end is containing SmaI restriction enzyme site, the other end is containing BstBI restriction enzyme site, with SmaI and BstBI restriction endonuclease simultaneously enzyme cut RFP fragment and pCDNA3.1 (+)-Panx3 recombinant plasmid, gel reclaims test kit and reclaims (health is ShiJi Co., Ltd), carry out two fragment connections, Transformed E .coli DH5 α competent cell with T4 DNA ligase.Test kit extracting pCDNA3.1 (+)-Panx3-RFP recombinant plasmid, restriction enzyme NcoI-HF enzyme is cut, and enzyme is cut product by 1.0% agarose gel electrophoresis qualification, electrophoresis result and in theory collection of illustrative plates identical (Fig. 2).Plasmid is delivered to Shanghai Sheng Gong company and check order, RFP gene sequencing result is correct.
The structure of embodiment 3 pCDNA3.1 (+)-Panx3-CMV-RFP
Taking pCDNA3.1 (+) as template, the amplification CMV promoter fragment taking primer 5 and primer 6 as primer PCR,
Primer 5:5 '-CGC
gGATCCgCTTGACCGACAATTGCAT-3 ', BamHI recognition site,
Primer 6:5 '-CGC
gGATCCaTTTCGATAAGCCAGTAAGCAG-3 ', BamHI recognition site.
PCR reaction conditions is: 94 DEG C of 3min, 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min totally 33 circulations afterwards, last 72 DEG C of 10min.
PCR obtains a linear DNA amplified production CMV fragment (732bp), two ends are respectively with BamHI restriction enzyme site, gel reclaims test kit and reclaims (health is ShiJi Co., Ltd), with BamHI simultaneously enzyme cut this fragment and pCDNA3.1 (+)-Panx3-RFP recombinant plasmid, carry out two fragment connections, Transformed E .coli DH5 α competent cell with T4 DNA ligase.Test kit extracting pCDNA3.1 (+)-Panx3-CMV-RFP recombinant plasmid, restriction enzyme NcoI-HF enzyme is cut, and enzyme is cut product by 1.0% agarose gel electrophoresis qualification electrophoresis result and collection of illustrative plates in theory coincide (Fig. 3).Plasmid is delivered to Shanghai Sheng Gong company and check order, CMV gene sequencing result is correct.
The structure of embodiment 4 pLL3.7-CMV-Panx3-CMV-RFP
NheI and PmiI double digestion for pCDNA3.1 (+)-Panx3-CMV-RFP recombinant plasmid; PLL3.7 lentiviral vectors is first used EcoRI single endonuclease digestion, re-use the quick end blunting of NEB test kit and make DNA end blunting, use again NheI single endonuclease digestion, gel reclaims test kit and reclaims (health is ShiJi Co., Ltd), carry out two fragment connections, Transformed E .coli DH5 α competent cell with T4 DNA ligase.Test kit extracting pLL3.7-CMV-Panx3-CMV-RFP recombinant plasmid, HindIII and NcoI enzyme are cut assay certificate and are obtained correct carrier (Fig. 4).This recombinant plasmid pLL3.7-CMV-Panx3-CMV-RFP is the red fluorescence lentiviral vectors of Panx3 gene overexpression of the present invention, and schematic diagram is as being shown in Fig. 5.
Embodiment 5 pLL3.7-CMV-Panx3-CMV-RFP are the impact that study on the carrier Panx3 gene pairs odontoblast discharges ATP function
One, experimental technique:
1. dentine like cell is grown up in pLL3.7-CMV-Panx3-CMV-RFP slow virus packaging and transfection
(1) according to 1 × 10
7individual/10cm culture dish carries out the inoculation of 293T cell, 37 DEG C of attachings of cell 24 hours.
(2) add three kinds of plasmids according to pMD2G, psPAX2, pLL3.7-CMV-Panx3-CMV-RFP order, quality is respectively 12,6,3 μ g, then adds serum-free opti-MEM substratum 1500 μ L, static 20 minutes of room temperature after thermal agitation.
(3) slowly add the mixing solutions of plasmid mixture and substratum to 293T cell surface, hatch 12 hours for 37 DEG C, change normal DMEM substratum, continue 37 DEG C and cultivate 24-48 hour.
(4) use 0.45 μ m strainer impurity screening, collect virus liquid.
(5) collect complete, healthy, fresh in vitro tooth and be put in containing in α-MEM substratum of 2% dual anti-(penicillin 500U/mL, Streptomycin sulphate 500 μ g/mL), transfer to rapidly Laminar Flow Room and carry out aseptic technique.First use the PBS of 2% penicillin-anti-streptomycin antibody to rinse dental surface, then the Exposed Pulp tissue of carefully tooth being rived, takes out pulp tissue under aseptic condition, is immersed in PBS, rinse out the sclerous tissues's fragment in pulp tissue, pulp tissue is transferred in α-MEM substratum and is cut into 1mm
3the tissue block of left and right, is inoculated into 25cm
2in plastic cell culture bottle, culture condition is α-MEM substratum, the 5%CO containing 20% foetal calf serum
2, cultivate in 37 DEG C of constant incubators.
Select third generation dental pulp stem cell, be prepared into single cell suspension after digestion, cell counting, according to 1 × 10
6the density in/hole is inoculated in 6 orifice plates, uses containing the α of 10% FBS-MEM and cultivates, and reaches 80% change mineralising inducing culture liquid (containing 50 μ g/mL xitix, 10mmol/L sodium β-glycerophosphate, 10 after converging until cell
-9mol/L dexamethasone), changed substratum 1 time every 3 days, cultured continuously 1 week, induces as becoming dentine like cell.
(6) inoculate into dentine like cell to 6 orifice plates, cell density is 2 × 10
5/ hole, while ensureing to infect, cell density reaches 90%.
(7) inhale and abandon substratum, virus liquid is slowly added drop-wise to cell surface, 37 DEG C leave standstill cultivation 6 hours.
(8) transfection, after 6 hours, is inhaled and is abandoned infection mixture, changes the DMEM substratum containing 10% serum, cultivates 48-72 hour for 37 DEG C.
(9) under fluorescent microscope, observation of cell infects rear red fluorescence expression, collects metainfective cell and adopts Real-time PCR and Western blot technology to observe transfection effect.
Collect the cell (crossing expression group) after normal odontoblast (control group) and virus infection, adopt Trizol to extract total RNA, according to reverse transcription test kit (TaKaRa) operation steps, be cDNA by RNA reverse transcription, carry out real-time fluorescence quantitative PCR as template.Taking house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference, adopt SYBR
rpremix Ex Taq
tMiI (TaKaRa) fluorescence dye kit measurement goal gene Panx3 relative expression content.Panx3 (171bp) primer: forward-CATCATCAGCGAACTGGAC, oppositely-TTACATGCCAGGTACCGCTCT; GAPDH (205bp) primer: forward-TTGTTGCCATCAATGACCCCTT, oppositely-GACTCCACGACGTACTCAGC.Gene specific upstream and downstream primer amplification system is: 95 DEG C of denaturations 4 minutes, and, then according to 95 DEG C of reactions 10 seconds, 60 DEG C of reactions 40 seconds, continue 40 and loop goal gene amplification.Panx3 gene relative expression quantity in cell RNA adopts 2
-△ △ CTcalculate, cross relative expression's difference multiple between expression group and control group and adopt fold change=2
-△ △ CTcalculate.
Adopt RIPA lysate lysing cell, and collecting cell lysate, use BCA protein determination kit to measure extracting solution protein concentration, carry out Western blot analysis.Application goat-anti people Panx3 monoclonal antibody (1:100, Santa Cruz company) and GAPDH (1:500, Santa Cruz) as primary antibodie, anti-as two with the anti-goat-anti body of rabbit (Beijing Zhong Shan Bioisystech Co., Ltd) of horseradish peroxidase HRP mark, ECL colour developing.
2. external stimulus discharges impact to ATP in odontoblast
(1) adopt ATP fluorescent marker dyes acrinamin (quinacrine) to observe ATP release conditions in cell, use fluorescence intensity analysis software Image Pro-plus to analyze ATP content in cell.Meanwhile, adopt bioluminescence technology to discharge ATP content to cell and measure, quantitative analysis stimulates ATP release conditions in lower odontoblast.Experiment is divided into 2 groups: control group, Panx3 cross expression group.
Control group: odontoblast.
Panx3 crosses expression group: pLL3.7-CMV-Panx3-CMV-RFP virus infection successfully becomes dentine like cell.
(2) in cell, ATP discharges observation
By 5 groups of cells according to 1 × 10
4/ hole density is inoculated in 24 orifice plates, after cell attaches completely, inhale and abandon substratum, use Krebs – Ringer – Hepes damping fluid (KRH:125mM sodium-chlor, 5mM Repone K, 1.2mM magnesium sulfate, 1.2mM dipotassium hydrogen phosphate, 2mM calcium chloride, 6mM glucose and 25mM Hepes/NaOH, pH=7.4) rinse cell 3 times, then every hole adds 50 μ M acrinamin KRH solution 1mL, 37 DEG C of lucifuges are hatched 30 minutes, use KRH damping fluid to rinse fluorescence dye, cell is placed under fluorescent microscope and is observed.Continue 2 groups of cells of record fluorescence intensity change situation in cell 0,5,10,15 second time after 4 DEG C of cold stimulations, and use Image Pro-plus to carry out fluorescence intensity calculating, using the ratio of the fluorescence intensity before each photo green fluorescence intensity and this group cytositimulation as fluorescence intensity reference value, to eliminate the difference of cell quantity and initial ATP content.
Two, experimental result
PLL3.7-CMV-Panx3-CMV-RFP virus infection odontoblast is after 48 hours, and under 558nm exciting light, odontoblast's cell is rendered as red fluorescence (Fig. 6), shows pLL3.7-CMV-Panx3-CMV-RFP transfection success.After pLL3.7-CMV-Panx3-CMV-RFP virus infection odontoblast, Panx3 mRNA and protein expression content are significantly higher than normal odontoblast group (Fig. 7).
Use ATP specificity fluorescent dyestuff poststaining cell, fluorescence intensity is directly proportional to intracellular ATP content.A is control group, and B is that Panx3 crosses expression group, and the fluorescence intensity after cold stimulation in control group odontoblast extends gradually and weakens in time; Panx3 crosses expression and organizes intracellular fluorescence intensity and reduce rapidly (Fig. 8).
Use Image Pro-plus to carry out fluorescence intensity calculating, the variation of ATP content in each group cell after cold stimulation, can find that ATP content reduces gradually in cell along with stimulation time extends, Panx3 crosses expression and organizes intracellular fluorescence intensity and underspeed and be significantly higher than control group (Fig. 9).
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan University
The red fluorescence lentiviral vectors of a <120> Panx3 gene overexpression and construction process and application
<130> 1
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 2711
<212> DNA
<213> Artificial Sequence
<220>
<223> Panx3-CMV-RFP gene fragment order
<400> 1
ctagcgttta aacttaagct tatgtcactt gcacacacag ctgcagagta catgctctca 60
gatgccctgc tgcctgaccg caggggaccc cgcctcaaag gactgcgtct ggaactgccc 120
ctggaccgga tagtcaagtt cgtagctgtg ggctccccct tgttgctgat gtccctggca 180
ttcgcccagg agttctcctc tgggtctccg atcagctgct tctctcccag taacttcagc 240
atccggcagg cagcctacgt ggacagctcc tgctgggact cactgcttca ccataagcag 300
gacgggcctg gccaggacaa aatgaaatct ctctggcccc acaaggccct cccctactcc 360
ctgctggccc tggccttgct catgtacctg ccggtgctgc tgtggcagta tgcagctgtg 420
ccagccctca gctccgatct gctgttcatc atcagcgaac tggacaaatc ttataatcgc 480
tccatccgcc tcgtgcagca catgctgaag atccggcaga agagttccga cccctatgtg 540
ttctggaatg agctggagaa ggctcggaaa gaacgatact ttgaattccc tttgctagag 600
cggtacctgg catgtaagca gcgttcacat tcgctagtgg ctacctacct cctgaggaac 660
tccctcttgc tcatcttcac ctccgccact tacctatacc ttggtcattt ccatctggat 720
gtcttcttcc aggaagaatt cagctgctcc atcaagacag ggctgctaag tgatgagacc 780
catgtcccca atctgatcac atgcaggctg acatcactgt ccattttcca gattgttagc 840
ctctccagtg tagcaatata caccatattg gttccagtga taatatacaa cctcacacgg 900
ctatgtcggt gggacaaacg acttttatct gtctatgaga tgctcccagc ttttgatctc 960
ctcagcagaa agatgctagg atgtcccatc aatgacctca atgtgatcct tcttttcctc 1020
cgagctaaca tctctgagct catctctttt agctggctga gtgtcttatg tgtgttgaag 1080
gatacaacca cccagaagca caatattgac acagtagttg attttatgac tttattggct 1140
ggcttagaac cctcaaaacc caaacacctc accaactcgg catgtgatga acacccatag 1200
ttaagaaacc atggagcaag aaagcttggt accgagctcg gatccgcttg accgacaatt 1260
gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat 1320
atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag 1380
ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct 1440
gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc 1500
caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg 1560
cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat 1620
ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca 1680
tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc 1740
gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga 1800
gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat 1860
tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctctctggc 1920
taactagaga acccactgct tactggctta tcgaaatgga tccaccggta gcatccgcca 1980
ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 2040
aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 2100
gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 2160
ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 2220
accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc 2280
gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg 2340
acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg 2400
taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 2460
gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 2520
ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 2580
tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 2640
aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taaacacgtg 2700
cggccgctcg a 2711
<210> 2
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 1
<400> 2
cccaagctta tgtcacttgc acacacag 28
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 2
<400> 3
cgcaagcttt ttgactcttc ggctcca 27
<210> 4
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 3
<400> 4
tcccccggga gacccaagct ggctagcg 28
<210> 5
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 4
<400> 5
cccttcgaac tggcaactag aaggcacagt cg 32
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 5
<400> 6
cgcggatccg cttgaccgac aattgcat 28
<210> 7
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> primer 6
<400> 7
cgcggatcca tttcgataag ccagtaagca g 31
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Panx3 forward primer
<400> 8
catcatcagc gaactggac 19
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Panx3 reverse primer
<400> 9
ttacatgcca ggtaccgctc t 21
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> GAPDH forward primer
<400> 10
ttgttgccat caatgacccc tt 22
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> GAPDH reverse primer
<400> 11
gactccacga cgtactcagc 20
Claims (5)
1. the red fluorescence lentiviral vectors of a Panx3 gene overexpression, it is characterized in that: taking lentiviral vectors pLL3.7 as skeleton carrier, after the CMV of pLL3.7 promotor, insert Panx3 gene, CMV promotor and RFP gene fragment according to 5 ' to 3 ' direction.
2. the red fluorescence lentiviral vectors of Panx3 gene overexpression according to claim 1, is characterized in that: the sequence of described Panx3 gene, CMV promotor and RFP gene fragment is as shown in SEQ ID NO.1.
3. the construction process of the red fluorescence lentiviral vectors of Panx3 gene overexpression claimed in claim 1, it is characterized in that comprising the steps: to design 5 ' holds the primer that contains restriction enzyme digestion sites, increase respectively Panx3 gene, CMV promotor and RFP gene fragment, be connected to Panx3 gene, CMV promotor and RFP red fluorescent protein fragment on pLL3.7 according to restriction enzyme site and the lentiviral vectors pLL3.7 restriction enzyme site of institute's amplified fragments 5 ' end.
4. the construction process of the red fluorescence lentiviral vectors of Panx3 gene overexpression according to claim 4, is characterized in that comprising the steps:
(1) extract total RNA of people pulp cells, taking the cDNA of reverse transcription as template, pcr amplification Panx3 gene fragment, the primer is:
Primer 1:5 '-CCCAAGCTTATGTCACTTGCACACACAG-3 ',
Primer 2: 5 '-CGCAAGCTTTTTGACTCTTCGGCTCCA-3 ';
By HindIII restriction enzyme site, Panx3 gene is linked and on pCDNA3.1 (+), obtained pCDNA3.1 (+)-Panx3 recombinant plasmid;
(2) taking Thy-Brainbrow-1.0H plasmid as template, pcr amplification RFP red fluorescent protein fragment, the primer is:
Primer 3:5 '-TCCCCCGGGAGACCCAAGCTGGCTAGCG-3 ',
Primer 4:5 '-CCCTTCGAACTGGCAACTAGAAGGCACAGTCG-3 ';
By SmaI and BstBI restriction enzyme site, RFP gene is linked and on pCDNA3.1 (+)-Panx3, obtained pCDNA3.1 (+)-Panx3-RFP recombinant plasmid;
(3) taking pCDNA3.1 (+) as template, pcr amplification CMV promoter fragment, the primer is:
Primer 5:5 '-CGCGGATCCGCTTGACCGACAATTGCAT-3 ',
Primer 6:5 '-CGCGGATCCATTTCGATAAGCCAGTAAGCAG-3 ';
By BamHI restriction enzyme site, CMV gene is linked and on pCDNA3.1 (+)-Panx3-RFP, obtained pCDNA3.1 (+)-Panx3-CMV-RFP recombinant plasmid;
(4) the Panx3-CMV-RFP fragment on pCDNA3.1 (+)-Panx3-CMV-RFP is linked to the red fluorescence lentiviral vectors that obtains Panx3 gene overexpression on pLL3.7 by NheI and PmiI restriction enzyme site.
5. the application of the red fluorescence lentiviral vectors of the Panx3 gene overexpression described in claim 1 or 2 in research Panx3 gene function.
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Cited By (3)
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| CN105063089A (en) * | 2015-05-19 | 2015-11-18 | 西安交通大学 | Method for immortalizing human exfoliated deciduous tooth endodontium stem cell line through hTERT recombinant lentivirus |
| CN106636166A (en) * | 2016-09-20 | 2017-05-10 | 中国水产科学研究院珠江水产研究所 | Recombinant plasmid pMDMcherry, construction method of recombinant plasmid and method for marking Edwardsiella ictaluri with red fluorescent protein gene |
| CN114990163A (en) * | 2022-03-31 | 2022-09-02 | 中海峡(福建)细胞生物科技有限公司 | Lentiviral vector for stem cell gene modification and construction method and application thereof |
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| CN103540616A (en) * | 2013-10-23 | 2014-01-29 | 中国人民解放军***福州总医院 | Lentiviral vector system for expressing diphtheria toxin A (DTA) fragment as well as preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063089A (en) * | 2015-05-19 | 2015-11-18 | 西安交通大学 | Method for immortalizing human exfoliated deciduous tooth endodontium stem cell line through hTERT recombinant lentivirus |
| CN106636166A (en) * | 2016-09-20 | 2017-05-10 | 中国水产科学研究院珠江水产研究所 | Recombinant plasmid pMDMcherry, construction method of recombinant plasmid and method for marking Edwardsiella ictaluri with red fluorescent protein gene |
| CN114990163A (en) * | 2022-03-31 | 2022-09-02 | 中海峡(福建)细胞生物科技有限公司 | Lentiviral vector for stem cell gene modification and construction method and application thereof |
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