定量PCR研究细粒棘球蚴抗氧化相关基因的差异表达  

The differential expression of the anti-oxidation related genes in Echinococcus granulosus by real-time PCR

作　　者：侯秋莲[1] 张富春[2] 张文宝[3] 吾拉木.马木提 张壮志[3] 

机构地区：[1]新疆医科大学基础医学院病原学教研室,乌鲁木齐830054 [2]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,乌鲁木齐830046 [3]新疆畜牧科学院兽医研究所,乌鲁木齐830000

出　　处：《中国人兽共患病学报》2010年第1期1-5,共5页Chinese Journal of Zoonoses

基　　金：美国国立卫生研究院R03基金资助项目(No.R03AI063367-01);国家自然科学基金项目(30760229);新疆教育厅创新群体资助项目(No.XJEDU2004G02)

摘　　要：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。To isolate the specific genes in protoscoleses（PSC）of Echinococcus granulosus under oxidative stresses from the SSH library constructed in the previous study,the gene expression in PSC under oxidative stresses was studied by using real-time PCR.The previously amplified library was sequenced and analyzed in GenBank with Blast research.Sequence analysis indicated that all clones in the SSH library contained the coding sequences,of which some clones showed homology in the GenBank and others were unknown.Differential expression of 4 genes randomly selected and the TPx gene in this library were studied with real-time PCR.It was demonstrated that the gene expression of S88 and H32-1 in oxidative tissues was 2.0 and 2.3 times higher than the un-oxidative stresses respectively.The TPx gene was up-regulated when PSC was induced with H2H2 of more than 0.8 mmol/L.These results implies that the up-regulated expression of the above-mentioned genes may be related with the related functions of anti-oxidative process in PSC and they may be used as the candidate genes for the study of anti-oxidation of E.granulosus.

关 键 词：细粒棘球蚴 氧化胁迫 抑制性消减杂交 荧光定量PCR 基因差异表达 

分 类 号：R383.33[医药卫生—医学寄生虫学]