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《響應(yīng)葡萄根瘤蚜侵染的擴(kuò)展蛋白基因篩選及其多克隆抗體的制備》由會(huì)員上傳分享,免費(fèi)在線閱讀,更多相關(guān)內(nèi)容在行業(yè)資料-天天文庫。
1、園藝學(xué)報(bào),2016,43(1):15–24.ActaHorticulturaeSinicadoi:10.16420/j.issn.0513-353x.2015-0574;http://www.ahs.ac.cn15響應(yīng)葡萄根瘤蚜侵染的擴(kuò)展蛋白基因篩選及其多克隆抗體的制備1211,*季興龍,韓寧,翟衡,杜遠(yuǎn)鵬12(山東農(nóng)業(yè)大學(xué)園藝科學(xué)與工程學(xué)院,作物生物學(xué)國(guó)家重點(diǎn)實(shí)驗(yàn)室,山東泰安271018;齊魯工業(yè)大學(xué)生物工程學(xué)院,濟(jì)南250353)摘要:以對(duì)葡萄根瘤蚜敏感的‘克瑞森無核’葡萄(Vitisvinifera‘CrimsonSeedless’)組
2、培苗和抗性砧木‘1103P’組培苗為試材,通過熒光定量PCR技術(shù)從擴(kuò)展蛋白基因家族中篩選到經(jīng)根瘤蚜侵染后表達(dá)上調(diào)的擴(kuò)展蛋白基因家族成員VvEXPA1。采用RT-PCR方法克隆到VvEXPA1基因片段,連接至原核表達(dá)載體pET-32a中,轉(zhuǎn)化大腸桿菌DE3菌株,測(cè)序確定構(gòu)建的重組載體pET32a-VvEXPA1開放閱讀框正確,并經(jīng)IPTG誘導(dǎo)表達(dá)了融合蛋白。該蛋白以包涵體的形式存在,純化后免疫新西蘭兔,制備擴(kuò)展蛋白多克隆抗體,通過Western-blot和ELISA檢測(cè)抗體的特異性及效價(jià)。結(jié)果顯示制備的抗體具有較高的特異性,ELISA顯示效價(jià)為
3、1︰51200。關(guān)鍵詞:葡萄;根瘤蚜;擴(kuò)展蛋白;VvEXPA1;多克隆抗體制備中圖分類號(hào):S663.1文獻(xiàn)標(biāo)志碼:A文章編號(hào):0513-353X(2016)01-0015-10IdentificationofVvEXPGenefromGrapeRootsInfectedbyPhylloxeraandPreparationofPolyclonalAntibodytoGrapeExpansin1211,*JIXing-long,HANNing,ZHAIHeng,andDUYuan-peng1(CollegeofHorticultureScienc
4、eandEngineering,ShandongAgriculturalUniversity,StateKeyLabofCropBiology,2Tai’an,Shandong271018,China;CollegeofBioengineering,QiluTechnologyofUniversity,Ji’nan250353,China)Abstract:BasedonqRT-PCRmethod,anexpansin(VvEXPA1)wasacquiredandinvestigatedfrominfectedCrimsonSeedlessg
5、rapeand1103Pgraperootstockinpresentstudy.VvEXPA1wasclonedintoprokaryoticexpressionvectorpET32aafteridentificationbyenzymaticdigestionandsequencing,andthentherecombinantplasmidwithVvEXPA1wastransformedintoE.coliDE3.TheresultsshowedthattheproteinwashighlyexpressedinE.coliandt
6、hemolecularweightoftherecombinantproteinwas41kD.Thespecificantibodywaspreparedbyimmunizingrabbitwiththepurifiedprotein,andthendetectedbyWestern-blottingandELISA.Finally,thehighspecificantibodywasobtainedandthetitersofanti-VvEXPA1antibodywere1︰51200.Keywords:grape;phylloxera
7、;expansin;VvEXPA1;poly-antibodypreparation收稿日期:2015–11–10;修回日期:2016–01–18基金項(xiàng)目:國(guó)家自然科學(xué)基金項(xiàng)目(31201609);山東省中青年科學(xué)家基金項(xiàng)目(BS2012NY007);山東省自然科學(xué)基金項(xiàng)目(ZR2013CQ022);高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金項(xiàng)目(20113702120007)*通信作者Authorforcorrespondence(E-mail:duyuanpeng001@163.com)JiXing-long,HanNing,ZhaiHeng,DuY
8、uan-peng.IdentificationofVvEXPgenefromgraperootsinfectedbyphylloxeraandpreparation