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ID:36623887
大小:2.46 MB
页数:105页
时间:2019-05-13
《家蚕生物反应器表达人乳铁蛋白及其功能研究》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
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
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