人酪氨酸酶相关蛋白┐2的cDNA克隆及表达.doc

2cDNA :-2; :-2TRP-2TRP-2.RNAcDNAPCRTRP-2pUC19GSTpGEX-4T-1IPTGTRP-2/GST.TRP-2cDNA;GSTpGEX-4T-1/TRP-2;TRP-2/GST.TRP-2GSTTRP-2/GST. Keywords:tyrosinaserelatedprotein-2;clone;fusionex-pression;vitiligo Abstract:AIMToclonethecDNAofhumantyrosinasere-latedprotein2TRP-2andexpressthecorrespondingprotein.METHODSTotalRNAwasisolatedfromculturedhu-manpigmentalcellsandmRNAwasreverslytranscribedintocDNA.ThenPCRwasusedtoamplifytheTRP-2codingre-gion.ThePCRproductwasclonedintopUC19plasmidandsequencedthensubclonedintovectorpGEX-4T-1.TheTRP-2proteinwasexpressedinE.coliofDH5asfusionproteinwithglutathioneS-transferaseGSTinducedbyIPTG.RESULTSTRP-2codingregionwasclonedintopUC19andthesequencewasconfirmed;FusionexpressingvectorofpGEX-4T-1/TRP-2wassuccessfullyconstructed;TRP-2/GSTfusionproteinwascorrectlyexpressed.CONCLUSIONHumanTRP-2geneissuccessfullyclonedandTRP-2/GSTfusionexpressingvectorisconstructed.TRP-2/GSTfusionproteiniscorrectlyexpressedinE.coli. 0 -2tyrosinaserelatedpro-tein-2TRP-2DHI-2.TRP-2TRP-2IgG.TRP-2TRP-2TRP-2.TRP-2cDNATRP-2. 1 1.1pUC19pGEX-4T-1DH5;RNA;BamHIEcoRIT4DNATaKaRa;TaqDNAPromega;GSTCloneTech. 1.2TRP-2.5BamHI3EcoRI.SBS:p15CG-GGATCC-ATGGGCTATAATTGTGGAGACTGCA3p25CG-GAATTC-GGCAAAGTTCCAGTAGGGCAAA3; RNAmRNAcDNA.PCR:952minTaqDNA1U9430s5530s7240s357210min.HPVTRP-1.PCR12g・L-1.BamHIEcoRIpUC19T4DNA.DH5BamHIEcoRI12g・L-1.:BamHIEcoRIpGEX4T-1DH5.TRP-2GST:0.4mmol・L-1IPTG37234hSDS-PAGE.1.:50LSDS-PAGE.ClotechGST. 2 2.1TRP212g・L-11457bpFig1.TRP-2pUC19BamHIEcoRI447bpFig2. 2.2TRP2/GSTGST2pUC19/TRP-2BamHIEcoRI447bpBamHIEcoRIpGEX-4T-1.DH5BamHIEcoRI447bpFig3GSTpGEX-4T-1/TRP-2. 14 pGEX-4T-1/TRP-2/DH537A6000.4234h.SDS-PAGEMr430002h4h.3100g・L-1SDS-PAGEFig5. 3 4.5.3-1TRP-16-2TRP-240%.TRP-2. TRP-2TRP-2TRP-2. 5 TRP-2438bp7211158bp-80%289294295300;146M r16990.RNART-PCRTRP-2GeneBankTRP-278.GSTpGEX-4T-1/TRP-2.IPTGlacTRP-2DH5TRP-2. : 1WuYMZhangJJJiZLLiuHPChenNCChenSM.CloningsequencingandexpressionofcDNAgeneofhumaneraJ.Di-siJunyiDaxueXuebaoJFourthMilMedUniv2000;215:646-648. 2ChengHLiuYFYuJYZhangSZZhangHZWangLG.Constructionandexpressionofanti-HCCimmunotoxinofsFv-TNF-andGFPfusionproteinsJ.Di-siJunyiDaxueXuebaoJFourthMilMedUniv2001;223:279-280. 3XiaoHSYangHJinWLHuangWJDuanXLChengBFJuG.Expressionandpurificationofrecombinanttenascin-RdomainsandtheirfunctionsJ.Di-siJunyiDaxueXuebaoJFourthMilMedUniv2000;2110:1182-1184. 4LiZQGaoTWZhaoXDChengJMLiuRQ.DetectionofautoantibodiestomelanocytesurfaceantigeninseraofpatientswithvitiligoJ.ZhonghuaPifukeZazhiChinJDermatol2001;344:268-270. 5LiZQGaoTWMaCLChengJMHuangHLiuRQ.De-tectionofautoantibodiestomelanocytesinvitiligopatientssseraandserainducedcytotoxicitymediatedbycomplementJ.LinchuangPifukeZazhiJClinDermatol2000;293:136-138. 6NishiokaEFunasakaYKondohHChakrabortyAKMishi-maYIchihashiM.ExpressionoftyrosinaseTRP-1andTRP-2inultraviolet-irradiatedhumanmelanomasandmelanocytes:TRP-2protectsmelanomacellsfromultravioletBinducedapoptosisJ.MelanomaRes1999;95:433-443. 7YanZWangJLHanWZhaoYTZhangYQSuCZ.CloningandexpressionofheatshockproteingeneofaHyper-thermophilicbacteriainE.coliJ.Di-siJunyiDaxueXuebaoJFourthMilMedUniv1999;207:587-588. 8WuGJHaoXKZhangYHBaiYJYaoLBJiSPLi