,2012,33(4):10-15 ProgressinVeterinary Medicine PGL3-insulinLuciferase * ( 361005) : PGL3-Basic 2 (insulin Ⅱ promoter) β MIN6,, : ; ; ; :Q782 :A :1007-5038(2012)04-0010-06,,, [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, 1 1.1 (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 ; :2011-10-31 : (1985-),, *
:PGL3-insulinLuciferase 11 50%, Lipofectine2000, 1.6 1.2 sense primer:ataacgcgtgtcgactct AGAGT- 1.2.1 PGL3-insulinluciferase 1.2.5 1.2.1.1 RenilaLucif- (PCR) 2 erase 10 1 β MIN6, (insulin Ⅱ promoter) PGL3- Basic,MluⅠ BglⅡ (D-Glucose,D-Glu) 1.0 4.5 9 22.5 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;4 1.2.1.2 MluⅠ BglⅡ PCR,, PGL3 700bp ( 1), 350ng/ μ L, PGL3,16 24 h,250 r/min 1.2.1.3 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 2 2.1 insulin Ⅱ promoter 10000 Rip-timer PCR DL2000;2~7. ;8.DNA 1.2.2 1.DNA MarkerDL2000;2-7.PCRproducts;8.DNA MarkerDL, MIuⅠ BglⅡ 10000, 1 PCR, NCBI Fig.1 ResultsoftargetgenePCR Blastn 2.2 1.2.3 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,,, 1 10 5 / 12
12 2012 33 4 ( 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 2.4.1 PGL3 MluⅠ BglⅡ ( 5), 4800bp 700bp, 2.4.2 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 DL10000 1.DNA MarkerDL2000 ;2-7.PCR products;;8.dna Marker DL10000 1.DNA DL10000;2~4.PGL3 ;5.DNA DL 1.DNA Marker DL 10000;2-4.PGL3-insulinluciferase;5.DNA MarkerDL2000 5 PGL3 Fig.5 IdentificationofPGL3-insulinluciferasebyenzymedigestion
: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,
14 2012 33 4 ( 226 ) A. Firelyluciferase ;B.Renilaluciferase ; C.FirelyLuc/RenilaLuc A.Glucose-inducedfirelyluciferasevalue;B.Renilaluciferasevalue; C.FirelyLuc/RenilaLuc 362:1090-1101. 7 [2] HeJ,GuD,WuX,etal.Majorcausesofdeathamongmen andwomeninchina[j].n EnglJMed,2005,353:1124-1134. Fig.7 EfectsofD-glucoseconcentrtiononexpressionofreportergene [3] WeyerC,BogardusC,MotD M,etal.Thenaturalhistoryof ( 7A), insulinsecretorydysfunctionandinsulinresistanceinthepath-, Renilaluciferase ogenesisoftype2diabetesmelitus[j].jclininvest,1999,, firelyluciferase 104:787-794. [4] OgohK,OhmiyaY.Biogeographyofluminousmarineostra- coddrivenirreversibly bythejapancurrent[j]. MolBiol ( 7B 7C), Renila, Evol,2005,22:1543-1545. [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,1999,58:749-757., 187-192. 3, 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,2002,268:160-168. [8] GhazawiI,CutlerSJ,LowP,etal.Inhibitoryefectsassoci- ated with use of modified Photinus pyralis and Renila reniformisluciferasevectorsindualreporterassaysandimpli- cationsforanalysisofisgs[j].jinterferon CytokineRes, 2005,25:92-102.
,2012,33(4):15-21 ProgressinVeterinary Medicine 1,2 2 2 2 1 2* (1. 450002;2., 510460) :, 11 807 PCR(nPCR) 1 (PCV-1) 2 (PCV-2); 2010 1 2011 8 PDNS 312 104, npcr PCV-2, PCR(RT-PCR), 11 5 PCV-1, PCV-2 PCV-2 30.61%, PCV-1 4.21%; 7 PCV ; PCV-2 58.65%,PRRSV 37.82%,PCV-2 39.89% PRRSV; 7 ~9 PCV-2, 1 ~3 ; PCV-2 27.9%,PRRSV 0.96%,PCV-2 PRRSV PCV PCV-2, PCV-2 PRRSV PCV-2, : ; :S852.659.2 :A :1007-5038(2012)04-0015-07 [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 ) :2011-11-21 : (2003B21404 20052490100 2006B20301013); (04002083); (06105311) : (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,1995,269:897-902. nessofthepgl3andpgl4luciferasereportervectorstoster- [11] SchaferSA,MachicaoF,FritscheA,etal.Newtype2diabe- oidhormones[j].biotechniques,2005,39:203-207. 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