论文部分内容阅读
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 study the differential expression of EgCamKⅡ and EgCavβ1, a member of calcium signaling pathway, at different developmental stages of echinococcus granulosus (echinococcosis, protocereocytic and adult). Methods The total RNA was extracted from the adult, original and secondary echinococcus granulosus germinal layers of Echinococcus granulosus respectively. The cDNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis kit. Based on the CamKⅡ gene sequence and Cavβ1 gene sequence design, The gene-specific primers were used to quantitatively analyze the CamKⅡ gene and Cavβ1 gene by using SYBR Green Ⅰ Real-time PCR and the standard curve established by plasmid DNA. The selected internal reference gene was β-actin. Results Real-time PCR showed that both CamKⅡ and Cavβ1 genes were highly expressed in the adult stage. The expression level of CamKⅡ gene in adult was 20 295 times that of the germinal layer and 1152 times that of the original germinal layer The expression level of Cavβ1 was 5 526 times higher than that of adult germinal layers and 636 times higher than that of cytoplasm germinal layer. The relative expression levels of two genes, SQ / Actin, were 13.4 and 1.9, respectively. By means of one-way analysis of variance between groups, the expression of both genes in adult stage was significantly different from that in other two developmental stages (P <0.05). Conclusion The expressions of CamKⅡ and Cavβ1, the target proteins of praziquantel, were significantly different at different developmental stages of Echinococcus granulosus. The expression of CamKⅡ and Cavβ1 may be related to the biological functions of different stages of development.