用表达性差异显示分析技术分离人胎肝组织选择性表达基因

ISOLATION OF SPECIFIC EXPRESSION GENE IN HUMAN FETAL LIVER BY REPRESENTATIONAL DIFFERENCE ANALYSIS

目的 :建立 4月龄人胎肝组织选择性表达基因EST库 ,为研究胚胎肝组织特异表达基因提供有力的工具。方法 :采用表达性差异显示分析 (representationaldifferenceanalysis,RDA)技术建立 4月龄人胎肝组织选择性表达基因EST库 ,并测定部分克隆核苷酸序列 ,以竞争PCR检测代表性基因的差异表达。结果与结论 :以珠蛋白家族基因为标志 ,所建差减cDNA文库中α 珠蛋白家族基因的表达频率较未差减cDNA文库增高 7倍 ,同时该文库也含有多种与肝生长密切相关的基因 ,表明RDA技术确是研究差异表达基因的有效手段 ,所建EST库富集胎肝特异表达基因 .部分克隆序列分析获得 2种未报道序列 ,其中一株含有丝 /苏氨酸激酶结构域 。

Aim:To isolate specific gene expressed in human fetal liver tissue and find out genes with important biological functions.Me thods :mRNA obtained from 4 monthes human fetus liver tissue was used as tester,and mRNA obtained from adult liver tissue was used as driver,and representational difference analysis(RDA) was performed.The subtracted final product was subcloned into the pGEM T easy vector,after transformation, bacterial colonies were randomly picked,and 54 plasmid clones with inserts were purified and sequenced.The homology search of genes was peformed by online based BLAST program through the nonredundant and EST database at Nation Center of Biotechnology Information(NCBI),the expression pattern of novel gene was further verified by competitive PCR.Results:Marker with α globin gene family, its expression frequency in subtractive cDNA pool is 7 times than non subtractive cDNA library,and the pool contains many genes closely relalive with liver growth.These show that RDA is an effective method to isolate gene differential expression.Through sequencing some clones,we get two sequences not reported before.Conclusion:The library is a useful means to study genes expressed specially in fetal liver.