日本血吸虫病肝纤维化小鼠肝星状细胞差异表达cDNA文库的构建和筛选

目的构建日本血吸虫病肝纤维化小鼠肝星状细胞差异表达基因的消减cDNA文库,并筛选差异表达基因。方法取日本血吸虫尾蚴经腹部感染小鼠致肝纤维化。采用抑制性消减杂交技术(SSH),分离小鼠肝星状细胞(HSC)及正常小鼠HSC,通过对比寻找差异表达基因。将其与T载体连接(T/A克隆),其产物转化大肠埃希菌DH5α,经文库扩增后,随机挑取白色克隆进行酶切鉴定,克隆经过正、反向杂交,阳性克隆经过测序和BLAST(局部相似性基本查询工具)进行表达序列标签(ESTs)差异基因分析。最后经模拟Northern印迹确认基因表达差异。结果扩增消减cDNA文库获得400余个白色阳性克隆,随机挑取的克隆经酶切鉴定后均有200～600 bp插入片段。ESTs分析获得76个序列,其中70个序列提示与血吸虫病肝纤维化或与其相关的基因,6个在公共数据库中未找到同源序列片段。结论用SSH法及T/A克隆技术成功构建了肝纤维化小鼠HSC与正常小鼠HSC差异表达基因的消减cDNA文库。

Screening of a Hepatic Stellate Cells Subtracted cDNA Library of Differentially Expressed Genes in Mice with Schistosomiasis japonica

OBJECTIVE: To construct and screen a hepatic stellate cells(HSCs) subtracted cDNA library, in order to seek differentially expressed genes in mice infected with Schistosoma japonicum. METHODS: HSCs were isolated from mouse as targets, and cDNA fragments of normal mouse were compared to those of S. japonicum infected mice to find dif ferentially expressed genes by technique of suppression subtractive hybridization(SSH). Differentially expressed cDNA fragments were then directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of DH5alpha. A subtracted cDNA library was constructed and then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for se quencing and BLAST analysis. Finally, virtual Northern blot confirmed such differential expression. RESULTS: The amplified library contained more than 400 positive bacterial clones. Random analysis of 100 clones with restriction enzyme digestion showed that all clones contained 200-600 bp inserts. 76 ESTs were obtained with 70 related to fibrosis caused by schistosomiasis or other etiological factors. Other 6 ESTs were not found in PubMed. CONCLUSION: The subtracted cDNA library of differentially expressed genes of HSC in normal mice and schistosome-infected mice was constructed successfully with SSH and T/A cloning techniques.