家蚕微孢子虫极管蛋白1(NbPTP1)的基因克隆及原核表达  

Cloning and Prokaryotic Expression of Nosema bombycis Polar Tube Protein 1(NbPTP1)

作　　者：吴玉娇[1] 龙梦娴[1] 陈洁[1] 李治[2] 李致宏[1] 潘国庆[1] 李田[1] 周泽扬[1,2] 

机构地区：[1]家蚕基因组生物学国家重点实验室,西南大学,重庆400715 [2]重庆师范大学生命科学学院,重庆400047

出　　处：《蚕业科学》2014年第2期258-264,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金重点项目(No.30930067);国家自然科学基金项目(No.31101770);高校博士学科点专项科研基金新教师类项目(No.20120182120014);中央高校基础研究项目(No.XDJK2012C085);西南大学博士基金项目(No.SWUB111081)

摘　　要：极管作为微孢子虫高度特异的结构,在微孢子虫侵染宿主细胞的过程中扮演重要角色。利用家蚕微孢子虫和兔脑炎微孢子虫基因组数据,通过共线性分析获得一个编码家蚕微孢子虫假定极管蛋白的基因NBO_7g0016。序列特征分析表明该基因编码富含脯氨酸的409个氨基酸,预测蛋白质分子质量39.6 kD,蛋白质序列的N端具有信号肽,有40个潜在的O-糖基化位点和17个磷酸化位点。以家蚕微孢子虫基因组DNA为模板克隆NBO_7g0016基因,并构建插入该基因的重组质粒,转化E.coli BL21(DE3)进行原核表达,融合蛋白经亲和层析纯化后免疫昆明小鼠制备多克隆抗体。免疫印迹实验证明该基因编码的蛋白质在成熟孢子中有表达,间接免疫荧光实验将蛋白质定位于家蚕微孢子虫的极管,确证该蛋白质为家蚕微孢子虫极管蛋白1(NbPTP1,GenBank登录号:EOB15198.1)。初步推测家蚕微孢子虫极管蛋白1具有糖基化修饰特征。Polar tube is a unique and highly specialized structure of the microsporidia. It plays a key role in the process of microsporidian invasion into host cells. Syntenic analysis of the genome between Nosema bombycis and Encepha/ito- zoon cunicufi led us to identify a hypothetic polar tube protein gene （NBO 7g0016） of N bombycis. Sequence feature analysis revealed that this gene encodes a protein of 409 amino acids rich in proline. It is predicted to have a molecular mass of 39.6 kD, a signal peptide at N-terminal, 40 O-glycosylation sites, and 17 phosphorylation sites. Using the ge- nome of IkL bombycis as template, NBO_7g0016 gene was cloned and constructed into prokaryotic expression vector pET32a（＋）. The recombinant plasmid was transformed into E. coil BL21 （DE3） competent cells for prokaryotic expres- sion. After purification with affinity chromatography, the fusion protein was used to immunize Kunming mice for preparingpolyclonal antibody. Western blotting analysis showed that the protein coded by this gene was expressed in mature spores of N. bombycis. Indirect immunofiuorescence assay （IFA） demonstrated that this protein was located in the polar tube. Therefore, this protein was confirmed as a polar tube protein of N. bombycis, named NbPTP1 （Gen- Bank accession No. EOB15198. 1 ） analysis suggested that NbPTP1 has the glycosylated protein. and preliminary characteristics of

关 键 词：家蚕微孢子虫 极管蛋白1 基因克隆 原核表达 免疫印迹分析 间接免疫荧光分析 

分 类 号：S884[农业科学—特种经济动物饲养]