P血型系統之分子基礎探討__臺灣博碩士論文知識加值系統

P血型系統是由醣脂質抗原組成的系統，然而其分子遺傳特性卻尚未釐清。

... 首先我們收集各100位血清型確認為P1與P2血型的捐血人並以PCR-RFLP法分析其A4GALT ... 資料載入處理中... 跳到主要內容 臺灣博碩士論文加值系統 ::: 網站導覽| 首頁| 關於本站| 聯絡我們| 國圖首頁| 常見問題| 操作說明 English |FB專頁 |Mobile 免費會員 登入| 註冊 功能切換導覽列 (167.99.71.17)您好！臺灣時間：2022/08/2212:06 字體大小：       ::: 詳目顯示 recordfocus 第1筆/ 共1筆  /1頁 論文基本資料 摘要 外文摘要 目次 參考文獻 電子全文 紙本論文 論文連結 QRCode 本論文永久網址: 複製永久網址Twitter研究生:王雅君研究生(外文):Ya-chunWang論文名稱:P血型系統之分子基礎探討論文名稱(外文):MolecularbasisofPbloodgroupsystem指導教授:林尊湄指導教授(外文):Tsun-MeiLin學位類別:碩士校院名稱:國立成功大學系所名稱:醫學檢驗生物技術學系碩博士班學門:生命科學學門學類:生物科技學類論文種類:學術論文論文出版年:2009畢業學年度:97語文別:中文論文頁數:77中文關鍵詞:流式細胞儀分析、P血型、A4GALT外文關鍵詞:Pbloodgroup、A4GALT、Flowcytometryanalysis相關次數: 被引用:0點閱:192評分:下載:13書目收藏:0 P血型系統是由醣脂質抗原組成的系統，然而其分子遺傳特性卻尚未釐清。

其中又以缺乏P1,PkandP三種抗原表現的p血型個體極為罕見，過去的研究認為是α(1,4)galactosyltransferase(A4GALT)活性缺失造成Pk抗原無法合成所致。

本研究第一個部份是探討一位臺灣p血型及其家族基因研究。

首先我們以polymerasechainreaction和直接定序分析A4GALT基因的codingregion，結果於9位家族成員中共有2位具homozygous核苷酸418和428之間呈現複合式剔除11bp暨插入34bp的突變現象，簡稱為418-428(del-ins)。

如此變異經由軟體預測會引發readingframeshift現象，而造成prematurestopcodon。

因此當我們以重組工程技術建構此突變型的construct並轉染於CHO細胞後，以flowcytometry與免疫螢光染色法僅見細胞微量表現Pk抗原。

此外，我們進行Galβ1-4Glc-PA轉移為Galα1-4Galβ1-4Glc–PA的酵素活性試驗並以HPLC醣類分析技術去測試A4GALT酵素活性，結果pmutant僅出現微量的波峰。

目前P血型系統的分子特性與決定P1/P2血型的主要調控基因仍是一大謎題，因此我們第二個研究部份是自A4GALT基因的codingregion多型性之間，尋找出調控P1抗原表現的相關因子。

首先我們收集各100位血清型確認為P1與P2血型的捐血人並以PCR-RFLP法分析其A4GALT基因109A>G、903C>G與987G>A。

依據初步的統計資訊中，我們自109A>G和987G>A的表現頻率去估測其所形成的haplotype頻率可以有意義地去區分P1與P2血型二個族群。

所以我們進一步地以PCR-SSP與直接定序去分析A4GALThaplotype109與987。

經重新統計分析發現，結果haplotypeGA與P1血型的表現具相關性(OR,9.33;95%CI,4.31-20.19)，然而haplotypeAG與AA傾向於P2血型的表現。

因此我們以定點突變建構兩種haplotypeAG與GA，並將帶有這兩種haplotype穩定性轉染的細胞株，以flowcytometry比較表面CD77的表現，結果發現haplotypeGA僅略高於haplotypeAG，因此目前仍不能完全證實haplotypeGA形成P1的表現型是因為有較高的A4GALT酵素活性才造成。

ThePbloodgroupsystemistheglycolipidantigensystemforwhichthemoleculargeneticbasishasnotyetbeenfullyillustrated.IndividualsofpphenotypeareveryrareandlackP1,PkandP,presumablyasaresultofdeficiencyintheenzymeα(1,4)galactosyltransferase(A4GALT),thatresponsibleforsynthesisofPkantigen.TheaimofthestudyistoexplorethemolecularbackgroundofafamilywithpphenotypeinTaiwan.ThecodingregionoftheA4GALTgenewasanalyzedbypolymerasechainreactionanddirectsequencing.Oftheninefamilyindividuals,thetwowithpphenotypearehomozygousforthecomplexmutationwithacombineddeletionandinsertionoccurredbetweenthenucleotide418and428;418-428(del-ins).Thisalterationresultinashiftinthereadingframeandaprematurestopcodon.Therefore,theminimalexpressionofPkwasshowedbyflowcytometryandimmunofluorescentmethodonCHOcellstransfectedwiththeconstructcontainingthecomplexmutation.Furthermore,carbohydrateanalysiswithHPLCwasperformedtoanalyzeA4GALTactivitybytransferingGalβ1-4Glc-PAtoGalα1-4Galβ1-4Glc–PA.ThepeakwasbarelydetectedinCHOcellstransfectedwithpmutantofA4GALTconstruct.ThemoleculargeneticbasisofthePbloodgroupsystemandtheP1/P2phenotypesdeterminationisstillunclear.ThesecondaimofthestudyistolookforpolymorphismsinthecodingregionofA4GALTgeneresponsibleforP1expression.WecollectedserologicallyconfirmedP1+(P1phenotype;n=100)andP1-(P2phenotype;n=100)blooddonorsandanalyzedthepolymorphisms109A>G,903G>Cand987G>AofA4GALTbythemethodsofpolymerasechainreactionandrestrictionfragmentlengthpolymorphism.Fromthepreliminarydata,allelefrequenciesfor109A>Gand987G>AestimatedhaplotypefrequenciesdifferedsignificantlybetweenP1andP2groups.ThenthehaplotypeofA4GALT109and987wasfurtheranalyzedbythemethodofPCR-specificsequenceprimeranddirectsequencing.TheresultsshowedthehaplotypeofGAwasfoundtobeP1-association(OR,9.33;95%CI,4.31-20.19),however,AGandAAwereP2-specific.Therefore,haplotypeofAGandGAatnucleotidepositionof109and987sitesofpcDNA4/V5/A4GALTweregeneratedbysitedirectedmutagenesis.ThestableclonesofhaplotypesAGandGAwereselectedtocomparetheA4GALTenzymeactivitybyflowcytometrytodetectCD77expressiononcellsurface.Nevertheless,theA4GALTactivitiesintransfectedcellswithhaplotypeGAwereonlylittlehigherthanhaplotypeAG.Therefore,itisstillnotconcludedhaplotypeGAwithhighenzymeactivityforP1expression. 中文摘要……………………………………………………………………….…….Ⅰ英文摘要…………………………………………………………………………….Ⅱ誌謝…………………………………………………………………………………..Ⅳ目錄…………………………………………………………………………………..Ⅴ表目錄……………………………………………………………...………………..Ⅷ圖目錄……………………………………………………………………………..…Ⅸ附錄圖表目錄…………………………………………………………….……….…Ⅹ縮寫簡索表……………………………………………..…………………….….....ⅩI儀器與藥品……………………………………………..…………………….…...ⅩⅡ第一章緒論…………………………………………………………………………1一、P相關血型系統…………………………………………………….……..1二、P相關血型系統的分子特性……………………………………………...1三、p血型與anti-PP1Pk……………………………………………………….3四、p血型與A4GALT基因變異的相關性……………………………….…..3五、調控P1/P2血型表現的P1基因…………………………………………4第二章研究動機……………………………………………………………………7第三章材料與方法…………………………………………………………………8一、P血型鑑定………………………………………………………………...8(一)檢體來源………………………………………………………………8(二)血清學方法測定P血型系統表現……………………………………8(1)紅血球凝集試驗試管法(tubemethod)……………………………8(2)管柱凝集法(columnagglutinationtest)………………………..…..9二、A4GALT基因分析………………………………………………………....9(一)GenomicDNA萃取……………………………………………………9(二)A4GALT基因的序列分析…………………………………………….10(1)A4GALT基因codingregion序列(-140)到+555………………..10(2)A4GALT基因codingregion序列386到1120……………………12(3)DNA定序…………………………………………………….…….13(三)利用PCR-RFLP(Restrictionfragmentlengthpolymorphism)區分A4GALT基因多型性………………………………………….……13(1)A4GALT109A>G基因多型性……………………………….……13(2)A4GALT903C>G基因多型性……………………………….……14(3)A4GALT987G>A基因多型性…………………………………….15(四)利用SSP-PCR(Sequence-SpecificPrimer-PolymeraseChainReaction)區分A4GALT基因多型性…………………………………………….16三、質體重組工程(Recombinantplasmidconstruction)……………………...18(一)聚合酶連鎖反應(PCR)………………………………..……………18(二)純化PCR產物………………………………………………………..20(三)質體接合(ligation)………………………………………………...…20(四)轉形(transformation)反應…………………………………………….22(五)小量質體萃取………………………………..……………………….22(六)大量質體萃取………………………………………………….……..23(七)定點突變………………………………………………….…….…….25四、細胞培養與轉染…………………………………………………….…….26(一)繼代細胞培養與轉染………………………………………….……..26(二)西方墨點法(Westernblotting)………………………………….……..29(1)SDS-PAGE分析…………………………………………….……...29(2)西方墨點轉漬法…………………………………………….………31(3)PCR…………………………………………………………………34五、A4GALT酵素活性分析…………………………………….…………….35(一)流式細胞儀分析(Flowcytometryanalysis)…………………………..36(二)免疫螢光染色法(Immunofluorescentstainingmethod)………...........37(三)Enzymeactivityassay…………………………………........................38(1)membranefraction萃取…………………………………................38(2)A4GALT酵素純化………………………………….......................39(3)A4GALT酵素活性試驗……………………………………………40(4)高效能液相層析儀系統(HPLC)………………………………..…40第四章結果………………………………………………………………………...42第五章討論………………………………………………………………………...46第六章參考文獻…………………………………………………………………...51表…………………………………………………………………………………….57圖…………………………………………………………………………………….63附錄………………………………………………………………………………….72 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