高凝状态大鼠肝脏差异表达基因反向消减cDNA文库的构建

目的构建高凝状态(prothrombotic states，PTS)大鼠肝脏差异表达基因反向清减cDNA文库．方法用高淀粉饲料喂养制备大鼠PTS模型，从PTS模型大鼠和对照大鼠肝脏提取poly A^ mRNA，分别以1．5 μg poly A^ mRNA起始，依次合成单链和双链cDNA，经酶切成平均大小为400-600 bp的片段。以PTS大鼠cDNA作为Driver，对照大鼠cDNA为Tester。将Tester分为两组，分别连上不同的接头，与Driver进行两次消减杂交和两次抑制性PCR。将第二次PCR产物cDNA克隆至pMD18-T载体上，然后转化细菌，转化后的细菌接种于含50 μg／ml ampicillin、IPTG、和X-gal的LB琼脂平板上，将克隆计数。结果获得的克隆78％为白色克隆，成功构建了PTS大鼠肝脏差异表达基因反向消减cDNA文库．结论以1．5 μg poly A^ mRNA起始，采用抑制性清减杂交，构建了PTS大鼠肝脏差异表达基因反向消减cDNA文库。

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

OBJECTIVE: To inquire into the mechanism of prothrombotic state (PTS) and the roles of liver therein by constructing a reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS. METHODS: The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS was constructed by suppression subtractive hybridization. The rat model of PTS was induced by a high-carbohydrate diet. Poly A+ mRNAs were isolated from PTS and control rats, and cDNAs were synthesized from the mRNAs. After digestion by means of Ras I, cDNAs 400-600 bp in size were obtained. For suppression subtractive hybridization, cDNAs from PTS rat were used as Driver and the cDNAs from control rat as Tester. The Tester was divided into two parts and ligated to adaptor 1 and adaptor 2R respectively. After two times of subtractive hybridization and two times of nested PCR, the products of the last PCR amplification were inserted into T/A plasmid vectors to transform the Escherichia coli JM109 cells. The transformed cells were incubated at 37 degrees C overnight on a LB agar plate containing ampicillin (50 micrograms/ml), IPTG and X-gal. The colonies were counted. RESULTS: 78% of the colonies were white and the reverse-subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state was successfully constructed. CONCLUSION: Prothrombotic state caused by malfunction of homeostasis and fibrinolysis is an important risk factor of cardiovascular disease. Liver plays important roles in the development of PTS, for the majority of the factors in the coagulation as well as fibrinolytic cascades are generated by the liver and secreted into the bloodstream. The reverse-subtracted cDNA library for differentially expressed genes in rat liver of PTS, successfully constructed in the present study, provides an efficient way to further investigate the mechanism of PTS and the relevant liver functions.