Cloning of Echinococcus multilocularis protein Musashi-1 and preliminary identification of its RNA-binding function
The aim of this study was undertaken to preliminarily investigate the distribution of Musashi-1 protein in Echinococcus multilocularis larvae and its ability to bind RNA.The full-length cDNA of E.multilocularis Musashi-1 gene was obtained by using RACE technology.This fragment of Musashi-1 was amplified and cloned into the pET-28a vector.The recombinant Musashi-1 protein was ex-pressed in Escherichia coli and purified,and prepared the polyclonal antibodies against Musashi-1.Immunofluorescence was employed to examine the distribution of Musashi-1 in E.multilocularis larvae.Additionally,the RNA-binding ability of Musashi-1 protein was evaluated using a dual-luciferase as-say.The results showed that there were two transcripts of the Musashi-1 gene of E.multilocularis,which were 1 017 bp and 966 bp,respectively.Western-blot analysis confirmed that the polyclonal antibody could specifically recognize the native E.multilocularis Musashi-1 protein.Immunofluorescence ex-periments indicated that the Musashi-1 protein is mainly located in the germinal layer cells of E.mul-tilocularis larvae.At the subcellular level,Musashi-1 protein is mainly distributed in the cytoplasm.A dual-luciferase assay demonstrated that the Musashi-1 protein could bind to GUA-rich RNA sequences recognized by Musashi family proteins.In this study,we determined the full length of the gene encoding Musasi-1 and confirmed that Musashi-1 is predominantly located in the germinal layer cells of E.multi-locularis larvae,and has the ability of RNA binding.These results aid to further investigate into the function of Musashi-1 in the germinal layer cells of E.multilocularis larvae.
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