組織型纖溶酶原源活劑(t-PA)cDNA克隆、定點突變及昆蟲細(xì)胞表達(dá).pdf

1、復(fù)旦大學(xué)博士學(xué)位論文組織型纖溶酶原源活劑(tPA)cDNA克隆、定點突變及昆蟲細(xì)胞表達(dá)姓名：劉軍波申請學(xué)位級別：博士專業(yè)：遺傳學(xué)指導(dǎo)教師：李寶健2000.4.1博士論文：組織型纖溶酶原激活荊(tPA)eDNA克隆、定點突變及昆蟲細(xì)胞中表達(dá)Cloningsitedirectedmutagenesisandexpressionininsectcellsoftissuetypeplasminogenactivator(tPA)cDNAAtpr

2、esent，thromboembolicdisordershavebecomeoneofthemaincausesofmoralityintheworldandtherearemorethanthreemillionpatientswithacutemyocardialinfarctionorcerebralthrombosisinourcountryTissue，typeplasminogenactivator(tPA)isnowth

3、oughiasthebestelinicthrombolvticagent，butithasnotyetdevelopedinchinaSotPAmustimportfromforeigncountries，butitspriceisveryexpensive，therefore，researchandproductionoft—PAwillhavesocialandeconomicvalueWiththeRT—PCRtechnique

4、tPAcDNAwasamplifiedfromhumanmelanomaBowescellsSequencingresultsshowedtlleclonedtPAcDNAsequencewassameasthatreportedbyPermicaetal～ThetPAcDNAt11atdidnotcontainedthesequenceencodingthesignalpeptideandprosequencewasexpressed

5、inEcoliTheexpressedtPA，whichhadfibrinolytieactivitywasobtainedafterpurifiedpreliminarilyThetPAexpressionininscctcellswasfirstreportedinourcountryInourworktPAexpressionlevelwas3103IU／h1，eg18lOIU／106cells，higherthanthatrep

6、ortedinotherexpressionsystemsThemolecularweightoftheobtainedtPAwasabout68kDa，similartothatofstandardtPApurifiedfromhumanmelanomaBowescellmediaComparedwithstandardtPAthestimulationby邱rinogen，affnitywithfibtinandinactivati

7、oninplasmaoftheobtainedtPAwerealmostsame，andthehalflifeofexpressedtPAinratbloodcirculationwasabout7min，almostsameasthatreportedbyforeignresearchersInordcrtoprolongtPAhalflifcresistinactivationbyinhibitors，increaseitsaffi

8、nitywithfibrinandfibrinolyticspecificity，eightmutationprimersweredesigned，synthesizedandusedtomutateeightsitesoftPAgene(L66A／Y67S，N117Q，Alal60_Ser”’_Asn“5一Trp2”，N184Q，R275E，del296—302，K416Y，A473S)onthebasisofresearchesab

9、outrelafionshivbetweentPAstructureandfunctionandwitllthehelpofproteinanalysissoftwareThetPAmutatedgenescontainingonetosevenmutations，respectively，wereconstructedwithsitedirectedmutagenesisteclllliqueThetPAgenecontainings

10、evenmutations(notincludingAlal60_Serl65一Asn248Trp2”mutatiomwasexpressedininsectcellsⅥ，llatevermethodwasusedthetPAactivitywasnotdetectedinthemediaofinsectcellsinfectedbyrecombinantvirusesThetPAmutantgene’whichcontainedfiv

11、emutationsanddidnotchangethetwoaminoacidsatthelocationof275and473，wasclonedintobaculovirustransfervectorpBacPAK9，apurerecombinantbaculovirusBacM0849PA6wasthenobtainedbylipofectinmediatedcotransfectionmethod，Very]arcfibri

12、nolyticactivitywasdetectedinmediaofinsectcellsinfcctedbytherecombinantviruswith$2390substratechromogenicmethodbutmorethall100timestPAactivitywasfoundinthesamemediawithfibrinagaroseplateassayAsaresult，itsfibrinolyticspeci

13、ficityincreasedmorethan100timesthanthatofstandardtPABecauseoftimeshortageandmethodrestriction，thehalflifeinbloodandresistancetoinhibitorsofM0849PAvariantwerenotidentifedAccordingtootherresearchers’works，theaminoacidmodif

14、icationsin67，117and184sitescouldprolongthetPAhalflifeandthedeletionof296302aminoacidscoulddecreasethetPAinactivationM0849PAvariantobtainedfromthisstudymayhavelongerhalflifethanwildtPAandresisttheinactivationbyinhibitorsb