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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发病机制打下良好的基础。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,经逆转录酶合成dscDNA,并经RsaⅠ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 isolate differentially expressed genes of Kaposi’s sarcoma (KS) and to establish corresponding cDNA libraries to lay a good foundation for revealing the pathogenesis of KS at the molecular level. Methods: KS sarcomas and normal skin tissues were collected from the same patient. Total RNA was extracted and dscDNA was synthesized by reverse transcriptase. The cDNAs were digested with Rsa Ⅰ and ligated with two different linkers. The normal tissues and tumor tissues were used as tester And driver for suppression subtractive hybridization (SSH). The genes differentially expressed in KS sarcoma and normal skin were initially screened. The differential gene was amplified by PCR and ligated into pGEM-Teasy cloning vector. The recombinant plasmid was transformed into E. coli DH5α. Blue and white screening, access to white positive colonies, and boiling broken bacteria, PCR amplification of unknown gene fragments. Results: The subtracted genes were mostly located at 200-500 bp in length, and two gene libraries representing the up-regulation and down-regulation in KS tumor tissues were successfully constructed. Conclusion: KS differentially expressed gene library was obtained by two-way suppression subtractive hybridization. Suppression subtractive hybridization is a rapid, convenient and effective method for establishing a differential gene library.