肾阴虚证cDNA文库的构建

Research on related genes of kidney yin deficiency syndrome

目的探讨肾阴虚证的相关基因。方法以辨病与辨证相结合,从糖尿病、慢性肾炎、狼疮性肾病以及亚健康状态等肾阴虚证入手,应用RNA微量扩增、抑制性消减杂交、基因克隆等技术和方法,分别构建肾阴虚证的cDNA子消减文库,再运用核酸分子杂交原理和PCR进一步筛选上述消减文库中的相同部分,以构建肾阴虚证的共同文库,然后将PCR产物与U载体连接,经蓝白斑筛选后,再用PCR方法插入片段筛选出阳性重组质粒。结果该研究成功地构建了肾阴虚证的cDNA文库,其中正向消减文库共获得625个阳性克隆,反向消减文库获得736个阳性克隆。结论该研究初步构建了肾阴虚证的cDNA文库,为进一步克隆肾阴虚证的相关基因奠定了基础。

[Objective] To research on the related genes of kidney yin deficiency syndrome from functional genomics. [Methods] RNA was isolated from white blood ceils using a commercial reagent RNA TRIzol. The RNA was then amplified by switching mechanism at 5＇-end of RNA transcript technique （SMART）. Purification of the amplified cDNA was used by spin-1000 purification column and the purified cDNA was digested by the use of Rsa Ⅰor Hae Ⅲ restriction enzyme. Comparing cDNA of experiment group （as Tester or Driver） with normal control cDNA （as Tester or Driver）, SSH was used to screen differentially expressed genes associated with kidney yin deficiency. After mirror orientation selection （MOS）, the PCR product was ligated with U plasmid vectors, transformed into E. coli TOP10, screened through the blue-white screening system. Then, positive recombinant clones were confirmed by PCR method. [Results] A total of 1361 clones were obtained： 625 from the forward subtractive library and 736 from the reverse subtracfive library. [Conclusion] The cDNA subtractive library was successfully constructed and it could provide a solid foundation for screening and cloning the related genes of kidney yin deficiency.