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目的在已构建的氧化胁迫下细粒棘球蚴(Echinococcus granulosus)与正常组织差异表达的消减cDNA文库中,筛选细粒棘球蚴在抗氧化过程中差异表达的重要基因。方法将前期研究中应用抑制性消减杂交技术(suppression subtrac-tive hybridization,SSH)构建氧化胁迫下细粒棘球蚴差异表达基因的消减cDNA文库进行蓝白斑筛选和菌落PCR分析后测序分析。测序结果利用BLAST在线软件与GenBank数据库进行同源序列比对分析和BlastX分析。从文库中随机挑选4个未知新序列和抗氧化密切相关的TPx基因片段,利用定量PCR法研究氧化胁迫下,差异表达基因片段在mRNA水平上的变化情况。结果整个文库克隆测序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。定量PCR结果显示:S88、H32-1两个基因在0.8mmol/LH2O2氧化胁迫的原头蚴中表达量分别上调为未经氧化胁迫原头蚴中的2.0和2.3倍,TPx基因片段当H2O2浓度大于0.8mmol/L时,其表达量增高。结论上述基因的上调表达很可能与细粒棘球蚴在抗氧化过程中的相关功能有密切的联系,可以作为研究细粒棘球蚴抗氧化的候选基因。
OBJECTIVE: To screen for the important genes that differentially expressed in hydatid cysts of Echinococcus granulosus during the oxidative stress induced by oxidative stress in subtracted cDNA libraries differentially expressed in Echinococcus granulosus and normal tissues. Methods Subtractive cDNA library of differentially expressed genes of Echinococcus granulosus under oxidative stress was screened by blue-white screening and colony PCR analysis using suppression subtrac-tive hybridization (SSH) in the previous study. Sequencing results Using BLAST online software and GenBank database homologous sequence alignment analysis and BlastX analysis. Four unknown new sequences were randomly selected from the library and related to the antioxidant TPx gene fragments, quantitative PCR was used to study the changes of mRNA expression of the differentially expressed genes under oxidative stress. Results The entire library was cloned and sequenced to obtain cDNA sequences of important genes such as oxidoreductase, protein kinase, growth factor and so on. Some other clones in GenBank can not find the corresponding homologous genes, may represent a new gene. The results of quantitative PCR showed that the expression of S88 and H32-1 genes were up-regulated respectively in 2.0 and 2.3 times of those in the absence of oxidative stress during the oxidative stress of 0.8 mmol / LH 2 O 2, respectively. When the concentration of H 2 O 2 More than 0.8mmol / L, its expression increased. Conclusion Up-regulated expression of the above genes is likely to be closely related to the related functions of hydatid cysts in the process of anti-oxidation, which can be used as a candidate gene for studying antioxidant activity of hydatid cysts.