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1、宁波大学硕士学位论文可口革囊星虫抗性基因的克隆与表达姓名：杜莉利申请学位级别：硕士专业：海洋生物学指导教师：李太武20090105宁波大学硕士学位论文编码区为327bp，开放阅读框长度为360bp，可编码120个氨基酸。从氨基酸序列来看，可口革囊星虫与库岛管体星虫的同源程度最高，与光裸方格星虫的同源程度次之。其理论蛋白质分子量为13.6294KD，理论等电点为5.78。经SDS-PAGE电泳分析，当IPTG终浓度为1mmol/L时，诱导4h时目的蛋白可以完全表达，重组菌在相对分子质量在14.3KD与20.1KD之间可见明显的条带。纯化后制备的
2、多克隆抗体效果理想。关键词：可口革囊星虫；热休克蛋白70；铁蛋白；蚯蚓血红蛋白；基因克隆和表达II宁波大学硕士学位论文CloningandExpressionofGenesRelatedtoResistanceofPhascolosomaesculentaAbstractInrecentyears,withtheindustrydevelopmentsuchasmetallurgy,instruments,printinganddyeing,heavymetalionssignificantlyincrease.Heavymetalnoton
3、lycausedpoisontomarine,butalsodepositedinmudofshallowseaarea.Ithadadverselyeffectonorganismswhichsurvivalandgrowthininter-tidalbeach.Phascolosomaesculenta,onekindofzoobenthos,isdeliciousandcontainsavarietyofprotein,aminoacidsandtraceelements.Soitisabletoadjustavarietyofbody
4、functionsandhavesomepharmacologicalactivities.ThatPhascolosomaesculentacangrowwellinthebadenvironmentcloselyrelatestoitisabletoresistbadlyconditionssuchaslow-temperature,high-temperature,hypoxia,heavymetalsandsono.SoPhascolosomaesculentahasveryimportantvaluetoberesearched.M
5、ergersprimersweredesignedbasedonthesequenceinformationofrelatedgenesreportedintheliterature.AseriesofmolecularbiologytechniquessuchasRNAextraction,RT-PCRandrapidamplificationofcDNAends(RACE)wereusedtoclonethegenesrelatedtoimmunityfromPhascolosomaesculentasuchasHSP70,ferriti
6、nandhemerythrin.Inaddition,therecombinantproteinwasexpressedinE.coliandpurified.Andthelast,detectionthespecificofpolyclonalantibodybyWesternblot.Theresultsobtainedareasfollows:Firstofall,thispapersuccessfullyaccessesthefull-lengthgenesequencesofHSP70fromthebloodofPhascoloso
7、maesculenta.Thisgeneisof2520bpbyincludinga5'untranslatedregion(UTR)of125bp,a3'UTRof421bpandanopenreadingframe(ORF)of1974bpencodingapolypeptideof658aminoacids.Theblastanalysisshowedveryhighhomology.TheestimatedmolecularmassofHSP70is71.5947KD,andthetheoreticalisoelectricpoint
8、is5.15.ThesecondarystructurepredictionthatHSP70isahybridprotein,whichprovidedstron
