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目的克隆鉴定细粒棘球绦虫原头蚴核糖体蛋白S9基因,分析其在细粒棘球绦虫不同发育阶段的表达情况。方法从已构建的cDNA文库挑选基因,克隆EgRPS9基因并对编码产物的理化性质、功能位点、蛋白质的结构和功能域进行预测和分析,通过RealTime-PCR检测RPS9基因在细粒棘球绦虫不同发育阶段的表达情况。结果通过各种生物数据库对EgRPS9基因进行预测并测序,显示该基因具有完整开放阅读框,大小为564bp,编码188个氨基酸,预测分子质量单位为22.0ku,等电点为10.32。SMART保守功能区域分析发现该基因第107~149个氨基酸为40S亚基S9结构域S4超家族。通过多序列比对发现该基因具有较高的保守性,进化树结果表明该基因与曼式血吸虫、日本血吸虫及华氏睾吸虫亲缘关系较近。Real time-PCR检测原头蚴和囊泡RPS9基因相对表达量分别为1.16和1.02,差异无统计学意义(P>0.05)。结论成功克隆出细粒棘球绦虫40S亚基RPS9新基因,该基因在细粒棘球绦虫不同发育阶段均有表达。
Objective To clone the S9 gene of the sporozoite antigen of Echinococcus granulosus and analyze its expression in different developmental stages of Echinococcus granulosus. Methods The genes of EgRPS9 were cloned from the constructed cDNA library and the physical and chemical properties, functional sites, protein structure and function domains of the encoded products were predicted and analyzed. RealTime-PCR was used to detect the expression of RPS9 gene in Echinococcus granulosus Developmental stage of the expression. Results The EgRPS9 gene was predicted and sequenced from various biological databases. The gene was found to have a complete open reading frame (ORF) of 564 bp in length and encoded 188 amino acids with a predicted molecular mass of 22.0 ku and an isoelectric point of 10.32. SMART conserved functional region analysis found that the gene 107 to 149 amino acids of the 40S subunit S9 domain S4 superfamily. The result of multiple sequence alignment showed that the gene was highly conserved, and the phylogenetic tree indicated that the gene was closely related to Schistosoma mansoni, Schistosoma japonicum and Fidaria. The relative expression levels of RPS9 gene in the progenitor cells and the vesicles were 1.16 and 1.02, respectively, by Real time-PCR, the difference was not statistically significant (P> 0.05). Conclusion The 40S subunit RPS9 gene of Echinococcus granulosus was successfully cloned and expressed in different developmental stages of Echinococcus granulosus.