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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 study the expression of EgAgB8 / 1, EgAgB8 / 2, EgAgB8 / 3, EgAgB8 / 4 and EgAgB8 / 5 in 8 stages of worm body, Echinococcosis germ layer, the original cercariae and adult) gene expression differences for the hydatid infection in the intermediate host and terminal host immunological diagnosis of screening phase specific dominant antigen. Methods The total RNA was extracted from the germinal layer of adult Echinococcus granulosus, egg, procidote and echinococcus respectively, and then reverse transcribed into cDNA using Avian Myeloblastosis Virus Reverse Transcriptase XL (TaKaRa, Japan). According to EgAgB 5 subunits (EgAgB8 / 1, EgAgB8 / 2, EgAgB8 / 3, EgAgB8 / 4, EgAgB8 / 5) sequences were designed to cross intronic gene-specific primers. Differences in gene expression were quantified using Real-ti me PCR using SYBR Green I and normalization of the quantification results with the actin II control gene. Results Real-time quantitative PCR results revealed that EgAgB8 / 1 and EgAgB8 / 2 were highly expressed in the germinal layer of echinococcosis (expressed in 68.874 and 16.258), while EgAgB8 / 3 and EgAgB8 / 5 were abundant in adult stage 1 905.512 and 180.315). The expression levels of EgAgB8 / 4 in adult, ovary and prototheca were 0.001, 0.088 and 0.102, respectively, which were lower than those in the germinal layer of hydatid cyst (3.576). Conclusion The gene expression of 5 subunits of EgAgB shows obvious difference in all stages of parasitism. EgAgB8 / 1 and EgAgB8 / 2 can be used as antigen candidates for immunological diagnosis of intermediate host of hydatid infection, while EgAgB8 / 3 can be used as target antigen for immunological diagnosis of terminal hydatid infection. The differential expression of the 5 subunits of EgAgB may be related to the biological functions unique to different stages of its development, which needs further study.