成人肝cDNA文库的构建  

Construction of Human Adult Liver cDNA Library

作　　者：苟德明[1] 郑新民[1] 陈乃清[1] 魏庆信 李文鑫 路兴中[1] 

机构地区：[1]西北农业大学动物科学系

出　　处：《中国兽医学报》1997年第5期449-452,共4页Chinese Journal of Veterinary Science

摘　　要：用异硫氰酸胍／盐酸胍法从人肝组织中提取ＲＮＡ，通过Ｏｌｉｇｏ（ｄＴ）纤维柱分离出ｍＲＮＡ。以ｍＲＮＡ为模板，含ＮｏｔⅠ接头的Ｏｌｉｇｏ（ｄＴ）为引物，在逆转录酶ＳｕｐｅｒＳｃｒｉｐｔⅡＲＴ催化下合成单链ｃＤＮＡ（ｓｓｃＤＮＡ），再在Ｅ．ｃｏｌｉＤＮＡ聚合酶Ⅰ与ＲＮａｓｅＨ和ＤＮＡＬｉｇａｓｅ共同作用下，ｓｃＤＮＡ拷贝成双链ｃＤＮＡ（ｄｓｃＤＮＡ）。经放射自显影测定，合成有全长ｃＤＮＡ片段。平末端ｄｓｃＤＮＡ与ＳａｌⅠ接头连接，ＮｏｔⅠ酶切后，通过过柱取掉小于５００ｂｐ的ｃＤＮＡ片段，大片段ｃＤＮＡ与载体ｐＳＰＯＲＴ１连接，转化受体菌ＤＨ５α，经稀释测定出含有重组子的文库大小为３．３×１０６。该文库适合于筛选低丰度ｍＲＮＡ的ｃＤＮＡ克隆。Total RNA was extracted from human adult liver tissue using Guanidnum thiocyanate/Guanidnum·HCl procedure. Poly(A) + mRNA was isolated and purified through packed column containing Oligo (dT) cellulose. The mRNA templates was reverse transcripted into single strand cDNA (sscDNA) with Not Ⅰ adapter Oligo (dT) primer catalyzed by SuperScriptⅡ RT. Second strand cDNA (dscDNA) synthesis was catalyzed by E.coli DNA polymeraseⅠ in combination with RNase H and DNA ligase. sscDNA was copied into dscDNA. It was proved by autoradiography that fully length cDNA was synthesised. Blund end dscDNA was ligased with SalⅠ adapter, and digested with NotⅠ. The cDNA fragments smaller than 500 bp was removed through column chromatography. The remaining large cDNA fragments was ligased with pSPORT1 plasmid vector, and then transformed to E.coli DH 5α . Thus, human adult liver tissue cDNA library contained 3.3×10 6 transformations, as determined on X Gal/IPTG plates, which is suitable to screen low aboundant mRNA for cDNA clone.

关 键 词：成人肝 CDNA文库 pSPORT1载体 

分 类 号：Q78[生物学—分子生物学]