高凝状态大鼠肝脏差异表达基因正向消减cDNA文库的构建与初筛  

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

作　　者：方定志[1] 刘秉文[1] 沈涛[1] 白怀[1] 

机构地区：[1]四川大学华西基础医学与法医学院生物化学与分子生物学教研室,成都610041

出　　处：《四川大学学报（医学版）》2005年第6期761-764,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家973计划(项目编号:G2000056900)资助

摘　　要：目的构建高凝状态(prothrombotic states,PTS)大鼠肝脏差异表达基因正向消减cDNA文库并进行初步筛选.方法从PTS模型大鼠和对照大鼠肝脏提取poly A+ mRNA,分别以poly A+ mRNA为模板,依次合成单链和双链cDNA,经酶切成400～600 bp大小的片段.以PTS大鼠cDNA作为Tester,对照大鼠cDNA为Driver,进行抑制性消减杂交.将消减杂交第二次PCR产物cDNA克隆至pMD18-T载体上,然后转化细菌,获得PTS大鼠肝脏差异表达基因正向消减cDNA文库.用巢式PCR扩增法制备'正向'和'反向'消减cDNA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正向消减cDNA文库进行筛选;将获得的阳性克隆cDNA进行序列分析;并与GenBank DNA 数据库中的DNA序列进行同源性分析.结果成功构建了PTS大鼠肝脏差异表达基因正向消减cDNA文库,初步筛选发现两条PTS差异表达cDNA片段.结论成功构建了PTS大鼠肝脏差异表达基因正向消减cDNA文库.Objective To construct and preliminarily screen the forward-subtracted cDNA library of differentially expressed genes in rat liver of prothrombotic state （PTS）. Methods The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization using cDNAs synthesized from mRNA of PTS rat as Tester and cDNAs from mRNA of control rat as Driver. The products from the last PCR amplification of suppression subtractive hybridization were inserted into a T/A plasmid vectors to transform the Escherichia coli JM109 cells. To produce the library, the transformed cells were incubated at 37 C overnight on a LB agar plate containing ampicillin（50μg/ml）, IPTG and X-gal. 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 The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed. Two differentially expressed cDNA fragments were found after preliminary screening. Conclusion The forward-subtracted cDNA library for differentially expressed genes in rat liver of PTS was successfully constructed in the present study.

关 键 词：高凝状态 大鼠模型 差异表达基因 CDNA文库 

分 类 号：R346[医药卫生—基础医学]