肾癌抑制性消减文库构建及差异表达基因鉴定

Construction and screening of suppression subtractive hybridization library of renal cell carcinoma

目的 应用抑制性消减杂交技术筛选人肾癌差异表达基因。 方法 以 786 0和HK 2为消减杂交对象构建人肾癌抑制性消减文库 ,挑选阳性克隆进行测序及GenbankBLAST分析。结果 文库包含 3 62个有插入片段的阳性克隆 ,随机分析 5 0个克隆 ,其中 2个为新基因 ,另 48个源于3 6个已知基因 ,这些差异表达基因与肿瘤细胞的转录、翻译、增殖、凋亡、代谢、信号转导、血管新生、膜受体表达异常等有关。 结论 该文库质量可靠 ,筛选出包括低峰度和新基因在内的肾癌差异表达基因 ,为进一步研究肾癌发生。

Objective To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC). Methods Poly A + RNA was isolated from RCC lines 786 O(tester) and renal cell(RC) lines HK 2(driver), respectively. SSH procedure was performed according to the protocol of the PCR Select cDNA Subtraction Kit (Clontech), and PCR products were cloned into pT Adv vector and transformed E.coli TOP10F′. All positive clones picked out were digested and some of which were sequenced. Results The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0 3 to 0 9 kb. Among 50 clones sequenced randomly, 2 represented unknown genes and the other 48 derived from 36 known genes. Conclusion The quality of the SSH library of human RCC is reliable and its construction is the basis for further screening differentially expressed genes of RCC.