:PGL3-insulinLuciferase 11 50%, Lipofectine2000, sense primer:ataacgcgtgtcgactct AGAGT PGL3-insulinluciferase RenilaLucif

Size: px

Start display at page:

Download ":PGL3-insulinLuciferase 11 50%, Lipofectine2000, sense primer:ataacgcgtgtcgactct AGAGT PGL3-insulinluciferase RenilaLucif"

韬进马
5 years ago
Views:

1 ,2012,33(4):10-15 ProgressinVeterinary Medicine PGL3-insulinLuciferase * ( ) : PGL3-Basic 2 (insulin Ⅱ promoter) β MIN6,, : ; ; ; :Q782 :A : (2012) ,,, [6-8] 9.7% 15.5% [1] 6000, (firelyluciferase) [2] (,renilaluciferase),, (insulinresistance,ir) (insulin Ⅱ promoter) (IGT ) 2 PGL3-Bas- ic [9], PGL3-insulinlucif- [3] erase β MIN6,, luciferase,, luciferase, MIN6, (luciferase,luc) PGL3-Basic Rip-timer ; ; β MIN6;PROMEGA ; [4], ; ;PRO- [5] MEGA Dual-Luciferase? Reporter AssaySystem; HycloneDMEM ;2.5g/L 1d~2d ;Gibco ;Lipofectine2000 ; : : (1985-),, *

2 :PGL3-insulinLuciferase 11 50%, Lipofectine2000, sense primer:ataacgcgtgtcgactct AGAGT PGL3-insulinluciferase RenilaLucif- (PCR) 2 erase 10 1 β MIN6, (insulin Ⅱ promoter) PGL3- Basic,MluⅠ BglⅡ (D-Glucose,D-Glu) Insulin Ⅱ promoter (pol- Rip-timer PGL3-Basic, ymerasechainreaction,pcr),, 1XPBS(pH7.4) MluⅠ BglⅡ, 300μLPromega TAGGG;Anti-senseprimer:AGAAGATCTGAGCTCT GAAGCAAGCACC PCR :94 5 min;94 1 min,53 1 min,72 1min,35 ;72 10min; MluⅠ BglⅡ PCR,, PGL3 700bp ( 1), 350ng/ μ L, PGL3,16 24 h,250 r/min PCR PCR PCR,94 5 min;94 1 min,53 1 min,72 1 min,35 1.DNA ;72 10min;4 (37,5% CO 2 ) 80%, μg/ 6h,, 45μg/ μ L 24h; 10min, 20 μl insulin Ⅱ promoter Rip-timer PCR DL2000;2~7. ;8.DNA DNA MarkerDL2000;2-7.PCRproducts;8.DNA MarkerDL, MIuⅠ BglⅡ 10000, 1 PCR, NCBI Fig.1 ResultsoftargetgenePCR Blastn β MIN6 β PGL3-Basic PCR MIN6, 150mL/L 5 MluⅠ BglⅡ ml/lβ- DMEM 2,PGL3 68.9ng/ μ L 18ng/ μ L 3,insulin Ⅱ promoter MluⅠ Bgl 1 2 Ⅱ 1.2.4,, MIN6,,, / 12

3 ( 226 ) 1,9.DNA DL2000;8,16.DNA DL10000;2~7.PGL3;10~15. 1,9.DNA MarkerDL2000;8,16.DNA MarkerDL10000;2-7.PGL3;10-15.Targetfragments 2 PGL3 Fig.2 DoubleenzymedigestionofPGL3andtargetfragments 2.5, ( MIN6 ), ( ), 3 PGL3-insulin Fig.3 PGL3-insulinluciferase 2.3 PCR PCR, PCR 1.DNA 4,PCR 700bp,, 4 PCR 2.4 Fig.4 ElectrophoresisofPCRproducts PGL3 MluⅠ BglⅡ ( 5), 4800bp 700bp, Blastn, ( 6A) Rip-timer insulin Ⅱ promoter ( 6B) Blastn, 3bp~601bp insulin Ⅱ promoter 74bp~672bp 2000 ( ) 66%( 6C), PGL3-insulinluciferase DL2000;2~7.PCR ;8.DNA DL DNA MarkerDL2000 ;2-7.PCR products;;8.dna Marker DL DNA DL10000;2~4.PGL3 ;5.DNA DL 1.DNA Marker DL 10000;2-4.PGL3-insulinluciferase;5.DNA MarkerDL PGL3 Fig.5 IdentificationofPGL3-insulinluciferasebyenzymedigestion

:PGL3-insulinLuciferase 13 A.Thesequenceoffragmentinobjectiveplasmid;B.ThesequenceofinsulinⅡ promoterinrip-timer;c.theblastnresultsofaandbinncbi A. ;B.Rip-timer insulin Ⅱ promoter ;C.

4 :PGL3-insulinLuciferase 13 A.Thesequenceoffragmentinobjectiveplasmid;B.ThesequenceofinsulinⅡ promoterinrip-timer;c.theblastnresultsofaandbinncbi A. ;B.Rip-timer insulin Ⅱ promoter ;C.A B NCBI Blastn 6 Fig.6 Thesequencealignmentresultsofbacterialsuspension 6h, MIN6, 1.0μg/ μ L 24h 1.0μg/ μ L( 5 mmol/l), 22.5μg/ μ L,MIN6, 9.0μg/ μ L 22.5μg/ μ L [10], 45μg/ μ L 1.0μg/ μ L MIN6 5 ( 7A) PGL3-Basic,, 22.5μg/ μ L,

5 ( 226 ) A. Firelyluciferase ;B.Renilaluciferase ; C.FirelyLuc/RenilaLuc A.Glucose-inducedfirelyluciferasevalue;B.Renilaluciferasevalue; C.FirelyLuc/RenilaLuc 362: [2] HeJ,GuD,WuX,etal.Majorcausesofdeathamongmen andwomeninchina[j].n EnglJMed,2005,353: Fig.7 EfectsofD-glucoseconcentrtiononexpressionofreportergene [3] WeyerC,BogardusC,MotD M,etal.Thenaturalhistoryof ( 7A), insulinsecretorydysfunctionandinsulinresistanceinthepath-, Renilaluciferase ogenesisoftype2diabetesmelitus[j].jclininvest,1999,, firelyluciferase 104: [4] OgohK,OhmiyaY.Biogeographyofluminousmarineostra- coddrivenirreversibly bythejapancurrent[j]. MolBiol ( 7B 7C), Renila, Evol,2005,22: [5] NaylorL H.Reportergenetechnology:thefuturelooksbright 3 PGL3-insulinLuciferase, Rip-timer insulin Ⅱ promoter PGL3-Basic 15 MluⅠ 36 BglⅡ, β MIN6 MIN6 1.0μg/ μ L,, MIN6,, β 22.5μg/ μ L,,,, β MIN6,, : [1] Yang W,LuJ,WengJ,Jia W,etal.Prevalenceofdiabetes amongmenand womenin China[J].N EnglJ Med,2010, [J].Biochem Pharmacol,1999,58: , , β [11] Ⅱ β Ⅱ, [6] ParsonsSJ,RhodesSA,ConnorH E,etal.Useofadual fireflyandrenilaluciferasereportergeneassaytosimultane- ouslydeterminedrugselectivityathumancorticotrophinrelea- singhormone1and2receptors[j].analbiochem,2000,281: [7] WangY,YuY A,ShabahangS,etal.Renilaluciferase-Ae- quoreagfp (Ruc-GFP)fusionprotein,anoveldualreporter forreal-timeimagingofgeneexpressionincelculturesandin liveanimals[j].molgenetgenomics,2002,268: [8] GhazawiI,CutlerSJ,LowP,etal.Inhibitoryefectsassoci- ated with use of modified Photinus pyralis and Renila reniformisluciferasevectorsindualreporterassaysandimpli- cationsforanalysisofisgs[j].jinterferon CytokineRes, 2005,25:

6 ,2012,33(4):15-21 ProgressinVeterinary Medicine 1, * ( ;2., ) :, PCR(nPCR) 1 (PCV-1) 2 (PCV-2); PDNS , npcr PCV-2, PCR(RT-PCR), 11 5 PCV-1, PCV-2 PCV %, PCV %; 7 PCV ; PCV %,PRRSV 37.82%,PCV % PRRSV; 7 ~9 PCV-2, 1 ~3 ; PCV %,PRRSV 0.96%,PCV-2 PRRSV PCV PCV-2, PCV-2 PRRSV PCV-2, : ; :S :A : (2012) [1-2] (Porcinecircovirus,PCV), 1974 [3], PCV-1 PCV-2 ConstructionofPGL3-insulinLuciferase PlasmidandFunctionIdentifiction KANG Yu-jia,MENG Ming-hui,L Zhong-xian (Schoolof PharmaceuticalScience,Xiamen University,Xiamen,Fujian,361005,China ) : : (2003B B ); ( ); ( ) : (1984-),,, * 櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙櫙 [9] DoughertyDC,SandersM M.Comparisonoftheresponsive- cretionin MIN6cels[J].AmJPhysiol,1995,269: nessofthepgl3andpgl4luciferasereportervectorstoster- [11] SchaferSA,MachicaoF,FritscheA,etal.Newtype2diabe- oidhormones[j].biotechniques,2005,39: tesriskgenesprovidenewinsightsininsulinsecretionmecha- [10] Ishihara H,AsanoT,TsukudaK,etal.Human GLUT-2 nisms[j].diabetesresclinpract,2011,93(s1):9-24. overexpressiondoesnotafectglucose-stimulatedinsulinse- Abstract:Diabetesisaveryseriousdiseaseandcausedbyinsulininsuficiency.So,insulingenetranscrip- tionishottargetonunderstandingortreatingdiabetes.here,weinsertedinsulin2promoter(insulinⅡ promoter)intoapgl3-basic-luciferaseplasmidandconstructedaninsulin-luciferasegenetranscriptionre- porter.thenwetransfectedthereporterplasmidintobetaisletcels MIN6andcheckeditsresponsefor glucoseinduction.wefoundthattheluciferaseactivitywasincreasedwithglucosestimulationatadose dependentmanner.theseresultssuggestedthat wesuccessfulyconstructedaninsulin-luciferasegene transcriptionreporterandprovidedapowerfultoolontheresearchininsulinsecretion. Keywords:diabetes;insulin;geneticrestructuring;dual-luciferasereportergene

μ μ - - β- - μ

SUA41 - β- - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - μ μ - - β- - μ μ - β- β- β- - - - - - - - - - - - - - - - - - - μ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -