虾肝肠胞虫(Enterocytozoon hepatopenaei)SWP2基因的克隆、表达及其在虾类病害检测中的应用  

Cloning,expression and application in detection of SWP2 gene in Enterocytozoon hepatopenaei

作　　者：沈卫锋[1] 郭琦 刘莉 牛宝龙[1] 翁宏飚[1] 楼宝 SHEN Weifeng;GUO Qi;LIU Li;NIU Baolong;WENG Hongbiao;LOU Bao(Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China)

机构地区：[1]浙江省农业科学院水生生物研究所,浙江杭州310021

出　　处：《浙江农业学报》2021年第6期993-1000,共8页Acta Agriculturae Zhejiangensis

基　　金：浙江省农业科学院新建学科经费。

摘　　要：虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)是虾类养殖过程中一种非常重要的病原微生物。目前常用的EHP检测方法是以核糖体18S基因为靶基因的PCR检测,但由于18S基因的高度保守性,该方法存在一定的非特异性扩增问题。本研究利用电子克隆方法,得到了特异性较高的EHP孢壁蛋白2(spore wall protein 2,SWP2)基因。该基因的cDNA全长为1019 bp,开放阅读框全长687 bp,编码229个氨基酸,预测相对分子质量为26.32 ku,5′非编码区为120 bp,3′非编码区为179 bp,系统进化分析表明与毕氏肠孢虫和绒螯蟹肠孢虫具有较近的亲缘关系。构建了重组表达载体pET15b-SWP2,利用原核表达得到SWP2蛋白,相对分子质量约为28 ku,并制备了该蛋白的单克隆抗体。采用常规PCR及Western blot方法对市售的商品虾进行了EHP感染情况检测,结果显示,两种方法均能准确地进行检测分析。本研究结果为虾农肝肠微孢子虫病的诊断提供了技术支撑,同时为SWP2蛋白功能的研究打下了基础。The Enterocytozoon hepatopenaei(EHP)from shrimp is a very important pathogenic microorganism in aquaculture.In present,the common method for EHP detection is PCR reaction which use ribosomal 18S gene as a target.The problem with this method is nonspecific amplification because of high conservatism of 18S gene.In this study,the spore wall protein 2(EHPSWP2)was cloned with silico cloning method.The length of the cDNA of the SWP2 was 1019 bp containing an open reading frame of 687 bp encoding a protein with 229 amino acids with predicted molecular weight of 26.32 ku,and 5’UTR is of 120 bp and 3’UTR is of 179 bp.The amino acids sequences and phylogenetic tree analysis showed that there is a close relationship among EHP,Enterocytozoon bieneusi and Hepatospora eriocheir.The recombinant expression vector pET15b-SWP2 was constructed for SWP2 protein expression,the molecular weight was 28 ku and the monoclonal antibodies against SWP2 protein were produced.The methods of PCR and western blot method were applied for EHP detection with Macrobrachium rosenbergii samples.This study provided a technical guarantee for the diagnosis of EHP disease by farmers,and laid the foundation for SWP2 protein research.

关 键 词：虾肝肠胞虫 SWP2基因 蛋白表达 单克隆抗体 病害检测 

分 类 号：S945.4+1[农业科学—水产养殖]