应用SSH技术研究H_2O_2胁迫下细粒棘球蚴基因的表达

目的构建H2O2胁迫下细粒棘球蚴(Echinococcus granulosus)与正常组织差异表达的消减cDNA文库。方法以H2O2胁迫细粒棘球蚴cDNA为试验方(tester),正常生长的细粒棘球蚴cDNA为驱动方(driver),应用抑制性消减杂交技术(suppression subtractive hybridization,SSH)研究H2O2胁迫下细粒棘球蚴基因的表达。结果文库扩增后得到124个阳性克隆,菌落PCR分析,均得到200~1000bp插入片段。将整个文库克隆进行测序,测得序列结果利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析。结果获得重要基因的cDNA序列,如氧化还原酶、蛋白激酶、生长因子等。另有部分克隆在GenBank中无法查到对应的同源基因,可能代表了新基因。结论成功构建了H2O2胁迫与正常组织差异表达的消减cDNA文库,为研究细粒棘球蚴在抗氧化过程中的相关靶基因筛选奠定基础。

Gene expression of Echinococcus granulosus under H2O2 stress using SSH technology

The gene expression of protoscoleces (PSC) of Echinococcus granulosus under H2O2 stresses was studied by using the suppression subtractive hybridization (SSH) technique, in which the cDNA from the materials treated with H2O2 solution was used as tester and the cDNA from the materials in normal growth as driver. The mRNA was isolated from the tester and driver respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. After test cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, the products were subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search. It was demonstrated that the subtractive library of genes regulated by H2O2 stresses was constructed successfully. The amplified library contained 124 clones, of which all clones had 200-1000 bp inserts. Sequence analysis indicated that 124 clones containing the coding sequences and some had homology in the GenBank with others. The obtained sequences may be target genes regulated by H2O2, that providing foundation for the further identify differentially expressed genes in PSC of E. granulosus exposed to oxidative stress.