高凝状态大鼠肝脏差异表达基因反向消减cDNA文库的初筛

Preliminary Screening of a Reverse-subtracted cDNA Library for Differentially Expressed Genes in Rat Liver of Prothrombotic State

目的 对本室构建的高凝状态 (PTS)大鼠肝脏差异表达基因反向消减 c DNA文库进行初步筛选。方法 用巢式 PCR扩增法制备“正向”和“反向”消减 c DNA探针 ;采用差异筛选方法 ,用这两种探针对 PTS大鼠肝脏差异表达基因反向消减 c DNA文库进行筛选 ;将获得的阳性克隆 c DNA进行序列分析 ;并与 Gen Bank DNA数据库 (non- redundant,and non- mouse and non- hum an EST entries)中的 DNA序列进行同源性分析。结果 差异筛选获得 5个阳性克隆 ,序列测定及同源性分析表明 ,其中 2个克隆很可能是 Gen Bank DNA数据库中尚没有的新基因或 DNA序列 ,另外 3个克隆很可能代表 Gen Bank DNA数据库中已有的基因或 DNA序列。结论 大鼠肝脏 5个PTS相关基因序列、尤其是其中 2个 Gen Bank中尚没有的新基因或新 DNA序列的发现 ,为深入探讨 PTS发生的分子机制及肝脏在

Objective To identify differentially expressed cDNA sequences for rat liver of prothrombotic state (PTS) by screening a reverse subtracted cDNA library. Methods Forward subtracted cDNA probes and reverse subtracted cDNA probes were prepared by nested PCR amplification, which were labeled with HRP. Positive clones were selected by differential screening in which forward subtracted and reverse subtracted cDNA probes were separately hybridized with the membranes slot blotted by plasmid DNAs amplified and isolated from the library. Inserts in the positive clones were submitted to DNA sequencing. Nucleic acid sequence homology search was performed against the GenBank DNA database (non redundant, and non mouse and non human EST entries) using the Standard nucleotide nucleotide BLAST [blastn] program via a network connection to the National Center for Biotechnology information. Results Five positive clones were found by differential screening. For 3 of the 5 sequences, known genes of high homology (Score>225 bits) in the GenBank DNA database were found. For the other 2 sequences, only genes of low homology (Score=56) or intermediate homology (Score=151) were found. Conclusion Five cDNA sequences were found PTS related. 3 of them may represent the genes that are either identical or nearly identical to previously reported genes The other 2 sequences are most probably new sequences not existing in GenBank DNA database, which have been registered in the GenBank DNA database as new sequences.