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ID:36801354
大小:2.52 MB
页数:63页
时间:2019-05-15
《基于聚乙烯二氨基三嗪的氢键型转基因载体》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、摘要本工作研制出一种新型的通过氢键作用于DNA碱基对以达到复合DNA目的非病毒载体。以自由基无规聚合的方式制备了聚乙烯二氨基三嗪(P、ⅢT)及其与乙烯基吡咯烷酮(VP)的共聚物,并精制出水溶部分。琼脂糖凝胶电泳显示PVDT基聚合物可有效复合DNA,并通过氢键作用使DNA缩合成密实状态,有效置换溴化乙锭(EB)。随着DNA的复合,圆二色谱(CD)发现DNA构象由B型向C型转变,并出现psi型谱线,可能缘于DNA高级结构的改变(长程手性结构)。透射电镜(TEM)下,被PVDT基聚合物缩合的DNA呈现球状、环状或大的聚集体,类似于阳离子载体缩合DNA的形态
2、。以荧光素酶和增强的绿色荧光蛋白报告基因研究PVDT基载体对非洲猴。肾(COS.1)细胞的转染,结果表明PVDT基载体具有满意的转染效率,比商品化ExGen500有着更高的转染活性和更低的细胞毒性。PVDT基载体可有效保护DNA免受DNaseI的酶解,复合物在牛血清蛋白(BSA)下仍具有高的稳定性。鉴于这些特点,PVDT基载体优于传统阳离子载体,并有望用于体内转基因应用中。关键词t聚乙烯二氨基三嗪;氢键;DNA缩合;基因转染;非病毒载体ABSTRACTAnovelgenetransfersystemcapableofcomplexingplasmid
3、DNA(pDNA)throughcomplementaryhydrogenbondingofguestswithbasepairswasconstructedinthiswork.Poly(vinyldiaminotriazine)(PVDT)homopolymeranditscopolymerswithpoly(1·vinyl-2-pyrrolidone)weresynthesizedviaconventionalradicalrandompolymerization,andwater-solublefractionswerecollected.T
4、hesePⅦBb部edpolymerswereshowntoefficientlycomplexpDNAanddisplaceethidiumbromidefEB)inEB—intercalatedpDNAsolutionsbecauseofthehydrogen-bonding—inducedconstrainedstateofpDNA.Itwasalsofoundthatuponcomplexation,pDNAseemedtoundergoB-cconformationchangeandcirculardichroismspectraassum
5、edapolymer-and··salt--induced(psi):typepatternthatWasrationallyascribedtOacertainchangeinthehi.gh-orderstructureofDNAcondensates.Transmissionelectronmicroscopypresentedseveralmorphologiesofspheresandtoroidswithinaggregates芝100nlil,resemblingDNAcondensationinducedbycationics.Int
6、ransfectionstudiesusingpDNA—encodingluciferaseorenhancedgreenfluorescentprotein,thissystemcouldefficientlytransfectCOS—lcells.ComparedtOthecommercialpolycationsystem,ExGen500,theseH-bondingvectorsdisplayedseveralmeritssuclIashighertransfectionefficiency,lowercytotoxicity,better
7、serulncompatibility,andstabilityinthepresenceofbovineserumalbumin(BSA).TheresultssuggestedthatPVDT-basedpolymersaresuperiortOpolycationcounterpartswithregardtotheirpotentialinvivoapplications.KEYWORDS:poly(vinyldiaminotriazine)(PVDT);hydrogenbonding;DNAcondensation;genetransfec
8、tion;nonviralvector独创性声明本人声明所呈交的学位论文是本人在导师指导下进行的研究工作和取
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