用抑制性差减杂交构建新疆Kaposi肉瘤差异表达基因文库

Construction of Differential Library of Xinjiang Kaposi′s Sarc oma by Suppression Subtractive Hybridization

目的:分离卡波氏肉瘤(Kaposi's sarcoma,KS)差异表达基因,并建立相应的cDNA文库,为从分子水平揭示KS发病机制打下良好基础.方法:采集KS肉瘤及来源于同一患者的正常皮肤组织,抽提总RNA,逆转录酶合成dscDNA,经Rsa I酶切,与2种不同的接头衔接,分别以正常组织和肿瘤组织作为tester和driver,进行双向抑制性差减杂交(suppression subtractive hybridization,SSH),初步筛选KS肉瘤与正常皮肤差异表达基因,将差异基因PCR扩增,产物与pGEM-Teasy克隆载体连接,转化DH5α大肠杆菌,采用蓝白斑筛选,获得白色阳性克隆菌落,并煮沸破菌,PCR扩增出未知基因片段.结果:差减得到差异基因片段多位于200～500bp,成功构建了2个分别代表在KS肿瘤组织中表达上调和下调的基因文库.结论:经双向抑制性差减杂交获得了KS差异表达基因文库.抑制性差减杂交是一种快速、方便、有效的建立差异基因文库的方法.

Objective： To screen differentially expressed genes in KS and construct the gene library to provide a new idea for clarification of the pathogenesis of the KS. Methods： Kaposi＇s sarcoma tissues and normal skin tissues were collected from the same patient. Total RNA was extracted and suppress subtractive hybridization （SSH） was conducted according to manufacture＇s manual. The positive control was provided by the kit in which foreign cDNA × 174DNA/Hae Ⅲ was added into the skeletal muscle cDNA. The positive control was performed in parallel with samples. Differentially expressed genes were linked with pGEM-Teasy cloning vector and transformed into E. coil DHSc~. The positive clones were screened by blue and white spot. PCR were used to amplify these genes. Results： Multiple differentially expressed genes strips located at 200 -500bp were obtained by suppression subtractive hybridization. Two differential libraries representing the up-regulated and down-regulated genes in KS were constructed. Conclusion： Differentially expressed genes in Kaposi＇s sarcoma differential libraries were gained, which will be a solid foundation to elucidate the pathogenesis of Kaposi＇s sarcoma.