【摘要】 目的:构建海南坡鹿外周血白细胞cDNA文库。方法:采用RNAiso Reagent(TAKARA)处理新鲜血液,以总RNA抽提试剂盒(上海华舜)制备总RNA。利用SMART(Switching Mechanismat5’end of RNA Transcript)技术,用PowerscriptTM反转录酶逆转录合成第一链cDNA,LD-PCR扩增获得cDNA双链,SfiⅠ酶切和CHROMA SPIN-400TM柱分级分离后,500bp以上的片段与pDNR-LIB载体连接,电转化入E.coliDH5α,建成原始文库。然后扩增文库并随机挑取单菌落,酶切鉴定重组子插入片段大小。结果:经鉴定,库容量达到1.9×106克隆,原始文库滴度为1.59×1010cfu/mL,重组率接近100%,扩增后的文库滴度为7.4×1012cfu/mL。插入片段大小分布为0.5～2.0kb,平均长度约为1.0kb。结论:所构建文库的各项指标均达到要求,为筛选海南坡鹿已知和未知功能基因提供了材料来源,且为进一步研究基因的结构和功能奠定了重要基础。更多还原

【Abstract】 Objective:A cDNA library from the peripheral blood leucocytes in Cervus eldi hainanus was constructed.Method:Total RNA was extracted from the peripheral blood leucocytes and the long-distance PCR(LD-PCR) method was used to amplify double-strand cDNA based on the SMART(Switching Mechanism at 5’end of RNA Transcript) techniques for construction of a full-length cDNA library.The PCR products were digested by proteinase K.After digestion with SfiⅠ and size fractionation using CHROMA SPIN-400TM columns,cDNAs(>500bp) were ligated to the SfiⅠ digested,dephosphorylated pDNR-LIB vector.The ligation mixture was transformed into E.coli DH5α.Then,the library was amplified,and clones were picked randomly from the library for screening size of cDNA inserts through enzyme digestion.Result:By electroporation,the primary constructed cDNA library contained 1.9×106 independent clones.The titer of the cDNA library was estimated as 1.59×1010cfu/mL,with the percentage of recombinant clones almost 100%,and that of the amplified library was 7.4×1012cfu /mL.The inserts varied from 0.5 to 2.0kb with average size was about 1 000bp.Conclusion:The Cervus eldi hainanaus cDNA library meets the requirement of a standard cDNA library.It could lay a strong basis for subsequent work of clone and selection of some kinds of fine functional genes.更多还原