细粒棘球绦虫EgM123基因序列分析及EgM家族基因在虫体不同发育阶段的差异表达分析

Analyses of EgM123 Gene Sequence and Differential Expression of EgM Gene Family in Different Development Stages of Echinococcus granulosus

对细粒棘球绦虫EgM123基因进行分子克隆和生物信息学分析,同时对EgM家族基因在虫体不同发育阶段的mRNA转录水平进行初步研究。采集绵羊肝脏包囊中原头蚴,提取总RNA,通过RT-PCR获取EgM123纯化的PCR产物,克隆到pGEM-T载体并测序,对测序结果进行结构和功能的预测分析。以Actin基因为内参基因,采用SYBR Green real-time RT-PCR技术,对不同发育阶段,即其原头蚴和成虫的EgM4、EgM9、EgM123的表达量进行检测。结果表明,EgM123基因ORF为594bp,编码197个氨基酸,蛋白分子质量为21.959ku,等电点为7.94。EgM123蛋白无信号肽、跨膜螺旋区和卷曲螺旋结构,是一种亲水性的可溶性蛋白质,存在23个磷酸化位点。根据亚细胞定位预测显示,EgM123蛋白主要位于细胞核内。二级结构预测含α-螺旋9.64%、β-折叠22.84%、无规则卷曲62.94%。EgM123氨基酸序列中有6个潜在的抗原表位。RT-qPCR结果显示,成虫阶段EgM家族基因表达量极显著高于原头蚴阶段。在发育的同一阶段,EgM123基因极显著高于EgM4、EgM9基因的表达量。通过EgM家族基因在虫体不同发育阶段的mRNA转录水平,EgM123基因在虫体成虫阶段高表达,以期为筛选细粒棘球绦虫终末宿主疫苗候选基因提供理论依据。

Molecular cloning and bioinformatics analysis of EgM 123 gene were performed, moreover, the expressions of EgM family genes in different developmental stages at the mRNA level of transcription of Echinococcus granulosus were detected in this study. The hydatid protoscoleces were collected from the o- vine livers with Echinococcus granulosus and the total RNA was extracted as a template. The amplification product of EgM123 gene was obtained by RT-PCR. Then the product was cloned into pGEM-T vector and sequenced. The results of sequence were analyzed to predict its structure and function. Using Actin gene as an internal control,the expressions of EgM4,EgM9 and EgM123 mRNA from the protoscoleces and the a- dults in the different stages were detected by qPCR. The results showed that the ORF of EgM123 gene contained 594 bp, encoding 197 amino acids. It had a molecular weight of 21. 959 ku, and the isoetectric point of 7.94. It is a hydrophilic soluble protein with 23 phosphorylation sites and without signal peptide, transmembrane helix region and a coiled coil structure. The EgM123 protein was mainly located in the nu- cleus by subcellular localization and secondary structure contained 9.64% of alpha helix,22.84% of beta helix and 62.94~//oo of random coil. The EgM123 had 6 potential antigenic epitopes by the indicated analysis of amino acid sequence. RT-qPCR results showed that the EgM family gene expression was significantly higher at the adult stage. The expression level of EgM123 gene was significantly higher than that of EgM4, and EgM9 genes in the same time period. EgM gene family was studied at the level of mRNA transcription in different developmental stages of the parasite,EgM123 gene is highly expressed in the adult stage,This study provided the theoretical basis on the candidate gene vaccine against Echinococcus granulosus in defin- itive hosts.