柔嫩艾美耳球虫延伸因子1α特性与功能初步分析

Preliminary Study on the Function of Elongation Factor 1αof Eimeria tenella

鸡球虫病在世界范围内对家禽业具有重大的经济影响,导致高死亡率、低增长、低生产力和高防治成本。由于球虫耐药性问题严峻,球虫病的防治重点倾向于研究方便安全,并且与活疫苗相比更容易生产的亚单位疫苗。因此,迫切需要新的方法来有效地控制球虫病,包括寻找特定的分子靶标。本文以柔嫩艾美耳球虫孢子化卵囊cDNA第一链为模板,扩增了延伸因子1α(Et EF1α)的ORF序列,生物信息学分析显示,该基因编码450个氨基酸,预测相对分子质量为49.1 ku,等电点为4.7;编码的蛋白无信号肽和跨膜结构域,但都含有N-肉豆蔻酰化位点。qRT-PCR和Western blot分析Et EF1α在虫体不同发育阶段的mRNA转录水平和蛋白翻译水平,结果显示,Et EF1α在子孢子(Spz)和裂殖子(Mrz)的mRNA转录水平高于未孢子化卵囊(UO)和孢子化卵囊(SO),翻译水平在UO和Mrz高表达;间接免疫定位发现,Et EF1α主要定位于Spz一端和整个Mrz,随着虫体在细胞内发育,荧光强度逐渐增强。分泌试验显示该蛋白为分泌蛋白,但不是由微线体分泌的。入侵抑制试验结果表明,兔抗r Et EF1α多克隆抗体能抑制子孢子入侵DF-1细胞,抑制率约为22%。综上表明,该蛋白可能参与了虫体在宿主细胞内的生长发育和子孢子入侵宿主细胞的过程。

Coccidiosis infected by Eimeria spp.has a major economic impact on the poultry industry worldwide,resulting in high mortality,low growth,low productivity,and high control costs.Drug resistance of Eimeria spp.is a serious problem with the widespread use of anticoccidial drugs.The focus of coccidiosis prevention and control tends to research on subunit vaccines that are convenient,safe,and easier to produce than live vaccines.Therefore,new methods are urgently needed to effectively control coccidiosis,including the search for specific molecular targets.In this study,cDNA of E.tenella sporulated oocysts(SO)as a template,we amplified the ORF sequences of the elongation factor 1αof Eimeria tenella(Et EF1α).Bioinformatics analyses shows that the gene encodes 450 amino acids with a predicted molecular mass of 49.1 kDa.The isoelectric points were 4.7.Sequence analysis found that the proteins encoded by Et EF1αhave no signal peptides and transmembrane domains,but contain N-myristoylation sites.The transcription and translation levels were analyzed using real-time quantitative PCR and Western blot.The results showed that the mRNA transcription levels of Et EF1αwere higher in the sporozoite(Spz)and merozoite(Mrz)than unsporulated oocysts(UO)and SO,and the translation levels were high in UO and Mrz.Indirect immunofluorescence localization showed Et EF1αwas mainly located at anterior of Spz and the whole cytoplasm of Mrz.As the parasites developed in the cell,the fluorescence intensity gradually brightens.The secretion assay indicated that Et EF1αwas a secretion protein,but not secreted from micronemes.Invasion inhibition assay showed that rabbit anti-r Et EF1αpolyclonal antibodies can inhibit sporozoite invasion of DF-1 cells.These results indicated that Et EF1αmay play a role in the growth and development of the parasite in the host cell and participate in the process of sporozoite invasion into the host cell.