虾肝肠胞虫极管蛋白3的鉴定

Identification of Polar Tube Protein 3 in Microsporidium Enterocytozoon hepatopenaei

为探明极管蛋白在EHP侵染宿主过程中的作用机制及虾肝肠胞虫病的预防和治疗提供新的靶标,通过克隆南美白对虾(Litopenaeus vannamei)肝肠胞虫(Enterocytozoon hepatopenaei,EHP)极管蛋白3基因(Polar tube protein 3,PTP3),并进行生物信息学分析,构建其核心区域部分序列编码的蛋白原核表达载体,诱导表达、纯化,制备多克隆抗体。结果表明,PTP3中最长的开放阅读框片段长3 390 bp,编码1 130个氨基酸,预测蛋白质分子质量为125 k D。该蛋白拥有13个N-糖基化位点和多个O-糖基化位点,未发现信号肽结构域;拥有117个磷酸化位点,其中苏氨酸磷酸化位点最多,为71个;二级结构较为简单,α螺旋占41.77%,是最大量的结构元件;延伸链占12.48%,无规卷曲占39.29%,β转角占6.46%。通过免疫印迹试验验证多抗的特异性,用免疫荧光实验观察极管蛋白在极管弹射时的状态,表明EHPPTP3分布于虾肝肠胞虫弹出的极管上,是新鉴定出的虾肝肠胞虫极管蛋白,确认了EHPPTP3的存在。通过生物信息学分析、序列特征、蛋白表达特征、多抗特异性以及定位特征证实本研究克隆的PTP3即为EHP极管蛋白3(EHPPTP3),是虾肝肠胞虫中新鉴定出的一种极管蛋白。

In order to explore the action mechanism of Enterocytozoon hepatopenaei(EHP) in the process of EHP infection and to provide a new target for the prevention and treatment of shrimp hepatocytosis,polar tube protein 3(PTP3) gene of EHP was cloned in Pacific white leg shrimp Litopenaeus vannamei and was analyzed by bioinformatics.The protein prokaryotic expression vector encoded by the sequence of the core region was constructed,and the expression was induced and purified,and polyclonal antibodies were prepared.It was found that the protein had the longest open reading frame of 3,390 bp in length and encoded 1,130 amino acids,which was predicted to have a molecular mass of 125 kD,with 13 N-glycosylation sites and multiple O-glycosylation sites,and without signal peptide domain.The protein had 117 phosphorylation sites rich(about 71) in threonine phosphorylation sites.In addition,these proteins had relatively simple secondary structure,and was composed of 41.77% helical region,which is the maximal structural elements of PTP3,12.48% extended strand and 39.29% random coil region,and only 6.46% of β-turn.The specificity of the polyclonal antibody was confirmed by Western blot,and the state of the polar tube protein during ejection was observed by immunofluorescence experiment,in which EHPPTP3 was distributed on the polar tube ejected by EHP.The fact that it was a newly identified polar tube protein of EHP was confirmed the existence of EHPPTP3.The homology comparison confirmed that PTP3 sequence of EHP in Pacific white leg shrimp was EHP polar tube protein 3(EHPPTP3),which is the newly identified polar tube protein in EHP through bioinformatics analysis,sequence characteristics,protein expression characteristics,multi antibody specificity and localization characteristics.