兴凯湖梅花鹿肝全长cdna文库的构建及鉴定.pdf

摘 要 本试验以兴凯湖梅花鹿肝脏组织为研究材料，用Trizol方法提取兴凯湖梅花鹿肝脏组 水解、纯化后，用Sill酶切；将酶切产物进行分级分离，回收400bp以上的cDNA组分， 并与xTriplEx2载体连接；连接产物经体外蛋白包装，产生原始文库；鉴定文库的滴度和 重组效率后，进行文库扩增．随机挑取96个噬菌斑，用载体克隆位点两端的通用引物进 反转录过程中采用附加序列的方法使全长mRNA得到反转录，所以得到的cDNA是完整 的，完全符合eDNA文库的要求．本试验成功构建了兴凯湖梅花鹿肝脏组织的cDNA文库。 兴凯湖梅花鹿肝脏组织表达型九噬菌体cDNA文库的成功构建，不仅保存了珍贵的梅 花鹿基因资源，而且对于梅花鹿中有明确功能的特异基因的克隆、表达以及新基因的克隆 与功能研究莫定了物质基础． 关键词兴凯湖梅花鹿，肝脏，cDNA文库 Abstract Tllctotal tissues RNAwas from lakeSikadeerliver TRIZOLmethod． separatedXmgkai usmg and usedfor SMART were first-strandeDNA PCRswere techniqueCOS]]I／3’primer synthesis．LD thenusedto double-swandthatwere the cDNAs then Sillandfractionated analyse by digested by CHROMASPIN-400columns．The cDNAswcmcollectedandJinkedto vector． longer400bp kTrip琅x2 Then)．phage rfActiolland were and packaging libraryamplificationperformed．Thcqualitiesoforiginal eDNA were libraries checkedconventionaltiterdetermination．Sixteen amplified strictly by plaques were andtested PCRwithuniversalderivedfromthe randomlypicked usmg primers sequenceflanking vector．Thetiter WaS the eDNA thetiterof Was ofprimarylibrary7．9×10pfu／mL amplifiedlibrary andtherateofrecombinantWasabove88％．Theinsertsize from 1．06x10pfu／ml ranged0．3～3．0kb． cDNA