海南坡鹿外周血白细胞cDNA文库的构建

Construction of cDNA Library from the Peripheral Blood Leucocytes in Cervus eldi hainanaus

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。

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-400^TM 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 1.9×10^6 independent clones.The titer of the cDNA library was estimated as 1.59×10^10cfu/mL,with the percentage of recombinant clones almost 100%,and that of the amplified library was 7.4×10^12cfu /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.