细粒棘球绦虫CamKⅡ及Ca_vβ_1基因在虫体发育过程中的表达  

Differences in expression of CamK Ⅱ and Ca_vβ_1 in Echinococcus granulosus in different stages of development

作　　者：刘欢元 李军[2] 王慧[2] 吴川川[2] 郭宝平[2] 张文宝[2] 王建华[1,2] 

机构地区：[1]新疆医科大学基础医学院,新疆乌鲁木齐830011 [2]新疆医科大学第一附属医院,新疆乌鲁木齐830054

出　　处：《中国病原生物学杂志》2015年第11期1002-1006,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31260272)

摘　　要：目的研究钙信号通路成员EgCamKⅡ基因及EgCavβ1基因在细粒棘球绦虫各发育阶段(棘球蚴生发层、原头蚴、成虫)差异表达。方法分别从细粒棘球绦虫的成虫、原头蚴以及棘球蚴生发层中提取总RNA,用RevertAid First Strand cDNA Synthesis kit反转录成cDNA,根据CamKⅡ基因序列及Cavβ1基因序列设计跨越内含子的基因特异性引物,用SYBR GreenⅠ实时荧光定量PCR(Real-time PCR)技术,通过由质粒DNA建立的标准曲线分别对CamKⅡ基因及Cavβ1基因进行定量分析,选用的内参基因为β-actin。结果 Real-time PCR显示CamKⅡ基因及Cavβ1基因均在成虫阶段高表达,CamKⅡ基因在成虫中的表达量是包囊生发层的20 295倍,在原头蚴的表达量是包囊生发层的1152倍;Cavβ1在成虫的表达量是包囊生发层的5 526倍,在原头蚴的表达量是包囊生发层的636倍。两基因的相对表达量SQ/Actin分别为13.4和1.9。通过组间单因素方差分析,两基因在成虫阶段的表达与其他两个发育阶段相比具有显著的差异(P<0.05)。结论吡喹酮的靶蛋白CamKⅡ基因及Cavβ1基因在细粒棘球绦虫各发育阶段的表达具有显著的差异,表达可能与虫体发育不同阶段特有的生物学功能有关。Objective To identify the expression of EgCamKl] and EgCav]31 in Echinococcus granulosus in different de- velopmental stages （the germinal layer of hydatid cysts, protoscoleces, and adult worms）. Methods Total RNA was i- solated from fresh samples of E. granulosus adult worms, protoscoleces, and the germinal layer （cells） of hydatid cysts. First stand eDNA was synthesized using the RevertAid First Strand eDNA Synthesis kit （Thermo Scientific）. In accord- ance with the gene sequence of EgCamK Ⅱ and the gene sequence of EgCaH31 , specific primers spanning the intron were designed. A recombinant plasmid harboring each gene was constructed and plasmid DNA was extracted. The level of ex- pression of EgCamK 11 and EgCaβl in different stages of development was quantified using SYBR Green I real-time PCR. Relative levels of gene expression were normalized to that of the house-keeping gene Egβ-actin. Results Real-time PCR results indicated that EgCamK Ⅱ expression was 20 295-fold higher in adult worms and 1 152-fold higher in protoseoleces than that in the cyst membrane. EgCa,β1 expression was 5 526-fold higher in adult worms and 636 fold higher in proto- seoleces than that in the cyst membrane. The relative levels （SQ/Actin） of EgCamK Ⅱ and EgCavβ1 expression in adult worms were 13.4 and 1. 9, respectively, and were significantly higher than levels of expression in other stages （P〈0.05）. Conclusion EgCamK Ⅱ and EgCa,β1, were highly expressed in adult worms, which may indicate that these genes play a role in the development of adult worms.

关 键 词：细粒棘球绦虫 钙信号通路 荧光定量PCR 基因表达 

分 类 号：R383.33[医药卫生—医学寄生虫学]