1份生产用中药蛇粗毒的DNA分子鉴定

:2007-10-22;:2007-12-25:中国人民解放军全军医药卫生科研基金(No.2006166125);广东省自然科学基金(No.06019716):陈念(1980-),男(土家族),湖北宜昌人,现为华南理工大学生物科学与工程学院博士研究生,硕士学位,主要从事中药分子鉴定研究工作.*:赵树进(1958-),男(汉族),山东黄县人,现任华南理工大学生物科学与工程学院博士生导师,博士学位,主要从事神经药理学研究工作.1DNA陈念1,2,赵树进2*,韩丽萍2(1.,510640;2.,510010):利用PCR技术鉴定干燥蛇粗毒的来源物种,并对基因组DNA的提取效果及提取过程中需注意的若干问题进行分析和讨论,同时还对若干针对不同长度目的基因扩增引物的使用效果进行验证从1份已保存了7年的生产用干燥蛇粗毒中提取总DNA,并以之为模板进行PCR扩增测序和序列分析使用该方法可从蛇粗毒中提取得到微量的总DNA,并成功扩增得到0.45~1.18kb不同长度范围的目的片段;测序结果提交NCBI,并与GenBank中的同源序列进行BLAST比对该蛇粗毒样品来自眼镜蛇,且极可能由多个不同的亚种或单倍型所组成该技术有可能推广用于对动物粗毒的来源进行准确和直接地鉴定:蛇粗毒;分子鉴定;线粒体基因;DNA测序:R282.73:A:1008-0805(2008)07-1578-03DNAMolecularIdentificationofOneSnakeCrudeVenomUsedforProductionCHENNian1,2,ZHAOShu-jin2*,HANL-iping2(1.DepartmentofBioscienceandEngineering,SouthChinaUniversityofSciencesandTechnology,Guangzhou510640,China;2.DepartmentofPharmacology,GeneralHospitalofGuangzhouMilitaryCommand,Guang-zhou510010,China)Abstract:ObjectiveToidentifydriedsnakevenombyPCR.MethodsToextractDNAandsubsequentsequencingofthemito-chondrialgenesfromasnakevenomthathadbeenpreservedfornearly7years.ResultsMinimalgenomicDNAcouldbeextractedfromthecrudevenombythismethod,andfromwhichdifferentlengthoftargetregionsthatrangedwithin0.45~1.18kbwereamplifiedsuccessfully.AllthesequencesweresubmittedtoNCBIandcomparedagainsttheirhomologoussequencesintheGen-Bankdatabase.ConclusionSequencealignmentanalysesapprovedthatNajanajawasitsmostprobableoriginalspecie,andthissamplecouldbeconsistedbysomedifferentsubspeciesorhaplotypes.Thisapproachoffersastraightforwardmethodfordirectandaccurateidentificationofanimalcrudevenoms.Keywords:Snakecrudevenom;Molecularidentification;Mitochondrialgene;DNAsequencing,,,()[1],,,,,,,,17DNA,11.1材料(:001212),2000-05,(Najanajaatra),41.2试剂与仪器DP303DNAMarkerIII(),2TaqPCRMasterMix(),PCR(),K(Amresco),(),dNTPs,PCR,rTaqDNADL15000DNA()PE-2400PCR(PerkinElmers),3730-XL96(ABI),GDS-8000(UVP)22.1蛇粗毒中总DNA的提取100mg,3ml5ml,,,10000g5min,,,,10000g1minDNA,:200lGA,,10l40mgml-1K,5630min,220lGB,6010min,220l,,GDPW,60l-7015782008197LISHIZHENMEDICINEANDMATERIAMEDICARESEARCH2008VOL.19NO.7,DNA,200l,DNA1.0%,0.8~10kb,2kb(1,2,30l)1(5-'3)'/kb16H1/16L1CTCCGGTCTGAACTCAGATCACGTAGGCTGACCGTGCAAAGGTAGCGTAATCACT0.45H43/L13GGGTGTCCAAAGAATCAGAAATAATCGGAGGCTTTGGAAACTG0.48Leu/ND4TACTTTTACTTGGATTTGCACCATGACTACCAAAAGCTCATGTAGAAGC0.90PPhe/PValAAAGCATAGCACTGAAAATGGAGTGCTTTGTTGTAAGCTAC1.00tRNATrpR/L4437bGGCTTTGAAGGCTCCTAGTTTCAGCTAAAAAAGCTATCGGGCCCATACC1.10H16064/L14910CTTTGGTTTACAAGAACAATGCTTTAGACCTGTGATCTGAAAAACCACCGTTGT1.182.2扩增反应和测序PCR50,lDNA0.6M(1)0.2mMdNTPs1PCR2UTaqDNA2TaqPCRMasterMix,,DNA,:943min,32~35,9430~60s,55~6060s,7260s,725minPCR5l1.0%PCR2.3洗脱方式和模板用量对扩增效果的影响(13DL15000(6MarkDNAMarkDNA))1DNA216H1/16L12.3.1洗脱方式的选择2,1~3(150,60ng30ng),45(35,lTE)0.45kb,,,DNAPCR,DNA1ng/l45,60l2.3.2最低模板用量MasterMix(1,12.5l)TakaRaTaq(2~4,12.5,6.25l3.125l)0.45kb(3),,,3.125l,PCR,3,100mg,60l,5l2.4不同长度目的基因的扩增效果14,2(16H1/16L1),3~8(5,l16H1/16L1,tRNATrpR/L4437b,H16064/L14910,H43/L13,Leu/ND4PPhe/PVal),,PCR(4,3),(35~40),DNA,,,,,,(6MarkDNA)(1MarkDNA)3DNA416H1/16L12.5序列提交和分析2.5.1序列提交GenBank,BLASTBioEditv7.0.5.3[2],2.5.2BLAST检索24,5PCR(GenBankEF413642EU181149),4TA,(EU181148)BLAST,16SrRNANajanaja(,Z46482.1)Najanajanaja(,L10674.1)Z46482.189%90%,L10674.186%88%;Z46482.1,97%2.5.3序列同源比对和相似性分析(,inpress),BioEdit(25):EF413642EU181149DNA,PCR,,1579LISHIZHENMEDICINEANDMATERIAMEDICARESEARCH2008VOL.19NO.72008197:,,,(EU181148),,,,,,,(EF413644EF413645),,,216SrRNA(:)DNADist(:)EF413642EU181149EU181148EF413645EF413644EF413642-0.02730.10310.07510.0695EU1811490.971-0.09730.06960.0641EU1811480.9030.910-0.02470.0297EF4136450.9250.9320.973-0.0098EF4136440.9320.9390.9710.987-516H1/16L13,,[3],,[4];,,,,[5];,,,,,,,[6]17DNA,:PCR,,;,DNABLAST,,,致谢:感谢广州军区广州总医院医学实验科在实验上给予的帮助!:[1],,.[J].,2003,3(14):108.[2]HallTA.Bioedit:auser-friendlybiologicalsequencealignmenteditorandanalysisprogramforWindows95/98/NT[D].NucleicAcidsSym-posiumSeries.London:OxfordUniversityPress,1999:95.[3]McLaneMA,SanchezEE,WongA,eta.lDisintegrins[J].CurrDrugTargetsCardiovascHaematolDisorders,2004,4(4):327.[4]FryBG,WinkelKD,WickramaratnaJC,eta.lEffectivenessofsnakeantivenom:speciesandregionalvenomvariationanditsclinicalimpact[J].JToxicolToxinRev,2003,22(1):23.[5]PowellRL,SanchezEE,PerezJC.Farmingforvenom:surveyofsnakevenomextractionfacilitiesworldwide[J].ApplHerpeto,l2006,3(1):1.[6]WusterW,GolayP,WarrellDA.Synopsisofrecentdevelopmentsinvenomoussnakesystematics[J].Toxicon,1997,35(3):319.15802008197LISHIZHENMEDICINEANDMATERIAMEDICARESEARCH2008VOL.19NO.7