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

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 ...

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

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 1 019 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.