家蚕生物反应器表达人乳铁蛋白及其功能研究

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1、浙江大学博士学位论文家蚕生物反应器表达人乳铁蛋白及其功能研究姓名：刘涛申请学位级别：博士专业：生物化学与分子生物学指导教师：张耀洲20051201总之，本研究成功的利用了家蚕杆状病毒表达载体系统高效表达了功能性重组人乳铁蛋白，有利于将来对人乳铁蛋白的进一步运用开发。关键词：人乳铁蛋白；家蚕幼虫；重组杆状病毒；抑菌作用：结肠炎IIAbstractHumanlactoferrin(hLF)isamultifunctionaliron-bindingglycoprotin．Inthisstudy,recombinanthumanlactoferrin(rh

2、LF)wasexpressedsuccessfullyusingBombyxmorinuclearpolyhedrosisbaculovirusexpressionsystem．Weclonedthel止FeDNAbyPT-PCRfromnormalhumanmammarygland．ABLASTsearchrevealedthattheN-terminalsequenceofrhLFwasdeterminedasGRRRRSVwhichwassimilartothatofthenativehLF,indicatingthecorrectproces

3、singofthesignalpeptideandidenticalposttranscriptionalprocessingofthesignalsequence．TherecombinantplasmidpBacPAK-hLFwasco-transfectedwithlinearizedBombyxmorinuclearpolyhedrosisvimsBm—BacPA_K6DNAintoBmNcells，thenhomologousrecombinationoccurredinsidethecells．TherecombinantvirusvBm

4、—hLFharboringhLFgenewaspurifiedbythreeroundofplaqueisolationandidentifiedbySouthernblotting．TherecombinantviruswasusedtoinfectBmNcellsandthelarvaeofsilkwormtoexpresstherhLF．TheimmtmoreactivitiesofexpressionproductswasdeterminedbyWesternblotting．Abandwithamolecularweightof～78kDa

5、wasfoundinbothBmNcellsandthesilkwormlarvae，lowerthanthatofthehLFstandard(82kDa)，whichmightbeduetoincompleteglycosylationorproteindegradation．Around13．5pgrhLFwaspurifiedfrom105BmNcellsinfectedwithvBm．hLFandtherhLFwasprovedtobebiologicallyactive．TheresultsindicatedthatrhLFandhLFs

6、tandardwerebacteriostatictoEcoli．andthebacteriostaticactivityofrhLFwassliglltlystrongerthanthatofhLFstandard．Itwasmostlikelyattributabletothedistinctgiycosylationwhichwereessentialtotheproteinfunctions．Inthisstudy，rhLFisexpectedtobeusefulasanaturalantibacterialmaterialingeneral

7、andclinicalfoods，drugs，andcosmetics．Furtherstudiesconcerningthemechanismofthisactivityarenowinprogress．AhighexpressionleveloffunctionalrhLFinsilkwormhemolymph(192mg／literofsilkwormhemolymph)wasreported．Theproductionleveloftherecombinantproteinwasmaximalataround120hpostinfection

8、andthendecreasedrapidly．n1DSS’inducedcolitis1sawell。es