多房棘球绦虫蛋白Musashi-1的克隆及RNA结合功能初步鉴定  

Cloning of Echinococcus multilocularis protein Musashi-1 and preliminary identification of its RNA-binding function

作　　者：吴易璇 仲顺虎 郭小腊 陈轶霞[1] WU Yixuan;ZHONG Shunhu;GUO Xiaola;CHEN Yixia(Life Science and Engineering College,Northwest Minzu University,Lanzhou 730030,China;Linxia Animal Disease Prevention and Control Center,Linxia 731100,China;State Key Laboratory of Animal Disease Control and Prevention/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州1730030 [2]临夏市动物疫病预防控制中心,甘肃临夏731100 [3]中国农业科学院,兰州兽医研究所,动物疫病防控国家重点实验室,甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2024年第10期1377-1384,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32260874)。

摘　　要：初步探究Musashi-1蛋白在多房棘球蚴中的分布及其与RNA结合的能力。利用RACE技术获得Musashi-1全长,扩增该基因并克隆到载体pET-28a上,表达并纯化Musashi-1重组蛋白用于制备多克隆抗体;免疫荧光观察Musashi-1在多房棘球蚴组织中的分布;双荧光素酶试验检测Musashi-1蛋白与RNA结合能力。结果显示多房棘球绦虫Musashi-1基因有两个转录本,分别为1017 bp和966 bp。Western-blot结果表明,制备的多克隆抗体能够特异性识别天然多房棘球绦虫Musashi-1蛋白。免疫荧光试验表明Musashi-1蛋白主要分布在多房棘球蚴生发层细胞中。在亚细胞水平上,Musashi-1蛋白主要分布在细胞质中。双荧光素酶试验表明Musashi-1蛋白能够与Musashi家族共识别RNA基序(富含GUA)结合。本研究确定了编码Musashi-1基因的全长,证实Musashi-1蛋白主要分布在多房棘球蚴生发层细胞中,具有RNA结合能力。本研究为进一步探讨多房棘球绦虫Musashi-1蛋白在多房棘球蚴生发层细胞中的功能提供了帮助。The aim of this study was undertaken to preliminarily investigate the distribution of Musashi-l protein in Echinococcus multilocularis larvae and its ability to bind RNA.The full-length cDNA of E.multilocularis Musashi-l 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 expressed 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-l protein was evaluated using a dual-luciferase assay.The results showed that there were two transcripts of the Musashi-l gene of E.multilocularis,which were 1017 bp and 966 bp,respectively.Western-blot analysis confirmed that the polyclonal antibody could specifically recognize the native E.multilocularis Musashi-l protein.Immunofluorescence experiments indicated that the Musashi-1 protein is mainly located in the germinal layer cells of E.multilocularis larvae.At the subcellular level,Musashi-l 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.multilocularis larvae,and has the ability of RNA binding.These results aid to further investigate into the function of Musashi-l in the germinal layer cells of E.multilocularis larvae.

关 键 词：多房棘球绦虫 RNA结合蛋白 MUSASHI-1 生发层 

分 类 号：S852.732[农业科学—基础兽医学]