运用抑制性消减杂交技术构建糖尿病肾阳虚证DNA消减文库

Suppression Subtractive Hybridization in Construction of DNA Subtractive Library of Kidney Yang Deficiency of Diabetic Mellitus

目的:运用抑制性消减杂交技术(suppression subtractive hybridization,SSH)构建糖尿病(Diabetic mellitus,DM)肾阳虚证DNA消减文库。方法:选择汉族中医辨证为肾阳虚证的DM患者以及正常人做对照组,进行正向和反向消减杂交。用RsaⅠ酶切用硅胶法抽提基因组DNA成大小不等的片段,分别连接两种不同的接头,进行两次消减杂交及两次抑制性PCR,将PCR产物与U/A载体连接,经蓝白斑筛选及菌液PCR筛选出阳性重组质粒,构建DM肾阳虚证DNA消减文库。结果:用SSH方法筛选出DM肾阳虚证的差异DNA片段,经克隆及PCR筛选出阳性重组质粒,从而成功地构建了DM肾阳虚证的DNA消减文库。结论:SSH技术能够快速有效地分离差异DNA片段以构建DM肾阳虚证DNA消减文库,为进一步筛选和克隆DM肾阳虚证相关基因奠定了基础。

Objective： This research constructed DNA subtractive library of kidney yang deficiency of diabetic mellitus （DM） by suppression subtractive hybridization （SSH） technique. Methods- SSH was used to obtain differentially genes associated with kidney yang deficiency of DM, which compared blood DNA of patients with kidney yang deficiency of DM （as Tester or Driver） to normal blood DNA （as Tester or Driver）. After digested by Rsa 1 enzyme restriction,Tester DNA were divided into two groups, which were ligated to the specific adaptor 1 and adaptor 2 separately. Then Tester DNA was hybridized with Driver DNA twice. After that differentially genes were obtained by twice of nested PCR ampliation. The PCR product was ligated with U/A plasmid vectors, screened through the blue-white screening system, and then positive recombinant clones were confirmed by PCR method. Results：The DNA subtractive library of kidney yang deficiency of DM was constructed successfully. Conclusion：The DNA subtractive library may provide the foundation for screening and cloning the related genes of kidney yang deficiency of DM.