Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the di- morphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization （SSH） was used to prepare a complementary DNA （cDNA） subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between dif- ferently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction （qRT-PCR）. It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.
目的:采用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建汉族人慢性肾炎(chronicglomerulonephritis CGN)肾阳虚证cDNA消减文库。方法:选择汉族人CGN且中医辨证为肾阳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Trizol一步法提取总RNA,用SMART(Switch Mechanism At 5 end of RNATemplate)技术逆转录并扩增总cDNA,用RsaⅠ酶切基因组cDNA成大小不等的片断,分别与两种不同的接头连接,进行2次消减杂交及两交抑制性PCR,建立抑制性消减杂交(suppression subtractive hybridization,SSH),在SSH基础上进行镜像选择(mirror orientation selection,MOS),将PCR产物与U载本连接,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CGN肾阳虚证消减文库。结果:用SSH方法筛选出了CGN肾阳虚证的差异cDNA片段,得到了443个白色克隆,再经PCR方法快速筛选出阳性重组质粒,从而成功地构建了CGN肾阳虚证的cDNA消减文库。结论:SSH技术能够快速有效地分离差异cDNA片段以构建CGN肾阳虚证cDNA消减文库,为进一步筛选和克隆CGN肾阳虚证相关基因奠定了基础。
