摘要
目的应用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建汉族人IgA肾病肾阴虚证cDNA消减文库。方法选择IgA肾病且中医辨证为肾阴虚证的患者以及正常人作为其对照组,进行正向和反向消减杂交。采用TrizolBD法提取总RNA,用SMART技术逆转录并扩增总cDNA,用RsaⅠ酶切基因组cDNA成大小不等的片段,分别与两种不同的接头连接,进行2次消减杂交及2次抑制性PCR,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,构建IgA肾病肾阴虚证消减文库。结果用SSH方法筛选出了IgA肾病肾阴虚证的差异cDNA片段,其中正向消减文库共获得325个阳性克隆,反向消减文库获得306个阳性克隆,从而成功地构建了IgA肾病肾阴虚证的cDNA消减文库。结论SSH技术能够快速有效地分离差异cDNA片段,成功构建了IgA肾病肾阴虚证的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 construct cDNA substractive library from kidney-yin deficiency patients with IgA nephropathy by suppression subtractive hybridization （SSH）. Method Total RNAs from normal and disease patients were isolated using Trizol BD. eDNA was produced by SMART RT-PCR. SSH was used to obtain genes associated with kidney-yin deficiency of IgA nephropathy by comparing blood eDNA of patients with kidney-yin deficiency of IgA nephropathy （as Tester or Driver） with normal blood eDNA （as Tester or Driver）. After Rsa Ⅰ enzyme digestion, Tester eDNA was divided into two groups for the ligation with the specific adaptor 1 and adaptor 2. Tester cDNAs were hybridized with Driver eDNA twice and then nested PCR twice. The PCR product was ligated with Uplasmid vectors, transformed into E.coli TOP10 and screened through the blue-white screening system. Positive recombinant clones were confirmed by PCR. Result cDNA subtractive library was successfully produced. 325 clones were obtained from the forward subtractive library and 306 clones were obtained from the reverse subtractive library. Conclusion SSH can be used for the rapid isolation of differentially expressed cDNA. The cDNA subtractive library may be used for cloning the genes involving kidney-yin deficiency in patient with IgA nephropathy.
出处
《热带医学杂志》
CAS
2009年第4期362-365,共4页
Journal of Tropical Medicine
基金
国家自然科学基金(No.30772691)
广东省科技攻关项目(No.2007B060401055)
高等学校博士学科点专项科研基金(No.20079981005)
关键词
IGA肾病
肾阴虚证
抑制性消减杂交
CDNA文库
IgA nephropathy
kidney-yin deficiency
suppression subtractive hybridization
cDNA subtractive library
