关键词: 日本血吸虫病, 寄生虫性肝疾病, 抑制性消减杂交, 基因表达

Abstract: 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 differentially 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 DH5α. 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 sequencing 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.

Key words: Schistosomiasis japonica, Parasitic liver diseases, Suppression subtractive hybridization, Gene expression