中国人兽共患病学报
中國人獸共患病學報
중국인수공환병학보
CHINESE JOURNAL OF ZOONOSES
2010年
1期
1-5
,共5页
侯秋莲%张富春%张文宝%吾拉木·马木提%张壮志
侯鞦蓮%張富春%張文寶%吾拉木·馬木提%張壯誌
후추련%장부춘%장문보%오랍목·마목제%장장지
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
細粒棘毬蚴%氧化脅迫%抑製性消減雜交%熒光定量PCR%基因差異錶達
세립극구유%양화협박%억제성소감잡교%형광정량PCR%기인차이표체
Echinococcus granulosus%oxidative stress%suppression subtractive hybridization%real-time PCR%dfferentially expressed gene
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 subtractive 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0.8 mmol/L H_2O_2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H_2O_2浓度大于0.8 mmol/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.
目的 在已構建的氧化脅迫下細粒棘毬蚴(Echinococcus granulosus)與正常組織差異錶達的消減cDNA文庫中,篩選細粒棘毬蚴在抗氧化過程中差異錶達的重要基因.方法 將前期研究中應用抑製性消減雜交技術(suppression subtractive hybridization,SSH)構建氧化脅迫下細粒棘毬蚴差異錶達基因的消減cDNA文庫進行藍白斑篩選和菌落PCR分析後測序分析.測序結果利用BLAST在線軟件與GenBank數據庫進行同源序列比對分析和BlastX分析.從文庫中隨機挑選4箇未知新序列和抗氧化密切相關的TPx基因片段,利用定量PCR法研究氧化脅迫下,差異錶達基因片段在mRNA水平上的變化情況.結果 整箇文庫剋隆測序結果穫得重要基因的cDNA序列,如氧化還原酶、蛋白激酶、生長因子等.另有部分剋隆在GenBank中無法查到對應的同源基因,可能代錶瞭新基因.定量PCR結果顯示:S88、H32-1 兩箇基因在0.8 mmol/L H_2O_2氧化脅迫的原頭蚴中錶達量分彆上調為未經氧化脅迫原頭蚴中的2.0和2.3倍,TPx基因片段噹H_2O_2濃度大于0.8 mmol/L時,其錶達量增高.結論 上述基因的上調錶達很可能與細粒棘毬蚴在抗氧化過程中的相關功能有密切的聯繫,可以作為研究細粒棘毬蚴抗氧化的候選基因.
목적 재이구건적양화협박하세립극구유(Echinococcus granulosus)여정상조직차이표체적소감cDNA문고중,사선세립극구유재항양화과정중차이표체적중요기인.방법 장전기연구중응용억제성소감잡교기술(suppression subtractive hybridization,SSH)구건양화협박하세립극구유차이표체기인적소감cDNA문고진행람백반사선화균락PCR분석후측서분석.측서결과이용BLAST재선연건여GenBank수거고진행동원서렬비대분석화BlastX분석.종문고중수궤도선4개미지신서렬화항양화밀절상관적TPx기인편단,이용정량PCR법연구양화협박하,차이표체기인편단재mRNA수평상적변화정황.결과 정개문고극륭측서결과획득중요기인적cDNA서렬,여양화환원매、단백격매、생장인자등.령유부분극륭재GenBank중무법사도대응적동원기인,가능대표료신기인.정량PCR결과현시:S88、H32-1 량개기인재0.8 mmol/L H_2O_2양화협박적원두유중표체량분별상조위미경양화협박원두유중적2.0화2.3배,TPx기인편단당H_2O_2농도대우0.8 mmol/L시,기표체량증고.결론 상술기인적상조표체흔가능여세립극구유재항양화과정중적상관공능유밀절적련계,가이작위연구세립극구유항양화적후선기인.
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 H_2H_2 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.
