细粒棘球绦虫抗原B8kDa亚单位在虫体发育过程中的阶段性表达研究  

Developmental stage-specific expression of Antigen B 8 kDa-subunits in Echinococcus granulosus

作　　者：张海涛[1,2] 陈璐[1,3] 马海梅[1,3] 马秀敏[1,3] 吾拉木.马木提 

机构地区：[1]新疆医科大学基础医学院病原学教研室,新疆乌鲁木齐830011 [2]梅州市嘉应医学院附属医院,广东梅州514031 [3]新疆医科大学第一附属医院包虫病重点实验室,新疆乌鲁木齐830011

出　　处：《新疆医科大学学报》2011年第3期231-235,共5页Journal of Xinjiang Medical University

基　　金：国家自然科学基金项目(30760229);科技部"863计划项目"(2007AA02Z411)

摘　　要：目的研究细粒棘球绦虫抗原B(EgAgB)8 kDa亚单位基因家族成员(EgAgB8/1、EgAgB8/2、EgAgB8/3、EgAgB8/4、EgAgB8/5)在虫体发育各阶段(虫卵、棘球蚴生发层、原头蚴及成虫)基因表达的差异,为包虫感染中间宿主和终末宿主的免疫学诊断筛选期特异性优势表达抗原。方法分别从细粒棘球绦虫成虫、虫卵、原头蚴和棘球蚴生发层提取总RNA,用Avian Myeloblastosis Virus Reverse Transcriptase XL(TaKaRa,Japan)反转录成cDNA,根据EgAgB 5个亚单位(EgAgB8/1、EgAgB8/2、EgAgB8/3、EgAgB8/4、EgAgB8/5)序列设计跨越内含子的基因特异性引物。基因表达差异应用SYBR Green I荧光实时定量PCR(Real-ti me PCR)技术进行定量分析,并用内参对照基因actin II来标准化定量结果。结果荧光实时定量PCR结果分析,EgAgB8/1和EgAgB8/2在棘球蚴生发层大量表达(表达量为68.874和16.258),EgAgB8/3和EgAgB8/5在成虫阶段有大量表达(表达量分别为1 905.512和180.315),EgAgB8/4在成虫、虫卵和原头蚴中的表达量分别为0.001、0.088和0.102,均较棘球蚴生发层表达量(3.576)低。结论 EgAgB 5个亚单位的基因表达在虫体发育各个阶段显现出明显差异。EgAgB8/1和EgAgB8/2可作为包虫感染中间宿主免疫学诊断最佳候选目的抗原,而EgAgB8/3可作为包虫感染终末宿主免疫学诊断的靶抗原。EgAgB 5个亚单位的阶段差异表达可能与其虫体发育不同阶段特有的生物学功能有关,有待进一步研究。Objective To understand expression profile of 8 kDa-subunits of Antigen B at different developmental stages of Echinococcus granulosus（E.granulosus） and select predominant target protein for immunodiagnosis of Echinococcus infection in intermediate hosts and definitive hosts.Methods Total RNA was isolated from fresh samples of E.granulosus adult worms,eggs,protoscoleces and hydatid cyst germinal layers,respectively.First stand of cDNA was synthesized using Avian Myeloblastosis Virus Reverse Transcriptase XL（TaKaRa,Japan）.Expression level of the target genes at different developmental stages of the parasite was quantified by SYBR Green I real-time PCR with sequence-specific intron-spanning primers.Relative gene expression results were normalized by parallel studies with internal control gene actin Ⅱ.Results The real-time relative quantification PCR results revealed that the gene expression level of EgAgB8/1 and EgAgB8/2 were up regulated in the hydatid cyst germinal layer（68.874 and 16.258）;EgAgB8/3 and EgAgB8/5 were over expressed in adult worms（1 905.512 and 180.315）;EgAgB8/4 was express at a relatively lover level in adult worms,eggs and protoscoleces（0.001,0.088,0.102） compared with that in metacestode germinal layer（3.576）.Conclusion Five subunits of EgAgB seemed to be differentially expressed at different developmental stages of the parasite;According to our data,EgAgB8/1 EgAgB8/2 may be the best choice of target antigen for immunodiagnosis of Echinococcus infection in intermediate hosts and the EgAgB8/3 for in definitive hosts.The stage-specific expression patterns of the gene family may relevant with different functions of this antigen during the parasite life cycle,this should be further evaluated in subsequent study.

关 键 词：细粒棘球绦虫 抗原B 荧光定量PCR 基因表达 

分 类 号：R383.3[医药卫生—医学寄生虫学]