柔嫩艾美耳球虫多肽:N-乙酰氨基半乳糖转移酶2的基因表达及转录动态分析  

Expression Profiling of Polypeptide:N-acetylgalactosaminyltransferase T2 during Eimeria tenella Developmental Cycle

作　　者：刘晓丽[1,2,3] 龚振兴 马雪婷[2,3] 曲自刚 韩政岚[2,3] 刘保红 袁如意[2,3] 杨孝朴 蔡建平[2,3] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室,兰州730046 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《畜牧兽医学报》2017年第11期2173-2180,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31272554);中国农业科学院农业科技创新工程专项(2014-LVRI-09)

摘　　要：蛋白质翻译后O-糖基化修饰普遍存在于真核生物、细菌和古细菌,多肽:N-乙酰氨基半乳糖转移酶2(polypeptide:N-acetylgalactosaminyltransferase 2,ppGalNAc-T2)是O-糖基化反应的限速酶。基因组数据分析揭示艾美耳球虫具有O-糖基化反应途径。为研究柔嫩艾美耳球虫(Eimeria tenella)蛋白质翻译后糖基化修饰及其关键酶的药靶效用,对Et ppGalNAc-T2基因进行了克隆,并检测其在E.tenella不同发育阶段的表达动态。根据EuPathDB中所预测Et ppGalNAc-T2基因序列设计引物,以RT-PCR方法扩增获得Et ppGalNAc-T2的ORF,插入酶切位点后连接到原核表达载体pGEX-6p-1进行诱导表达。提取E.tenella广东株未孢子化卵囊、孢子化7h卵囊、孢子化卵囊、子孢子和第二代裂殖子5个发育阶段的总RNA,以qRT-PCR法检测Et ppGalNAc-T2转录水平的动态变化。结果表明,Et ppGalNAc-T2的全长ORF为1 983bp,编码660个氨基酸,在E.coli Transetta(DE3)可溶性表达。qRT-PCR结果显示,Et ppGalNAc-T2转录水平随发育阶段不同而有显著差异,子孢子阶段表达水平最高、而在孢子化卵囊则几近不能检出。结果为进一步研究EtppGalNAc-T2的功能提供了试验基础。O-linked glycosylation is a form of protein posttranslational modifications that occurs in the Golgi apparatus of eukaryotes, and archaea as well as bacteria. Polypeptide. N-acetylgalac- tosaminyltransferase 2 （ppGalNAc-T2, EC 2.4.1.41） is the first key enzyme in the pathway of O-glycan biosynthesis. Whole genome sequencing of Eirneria spp has ever revealed that these protozoa possess the pathways for O-glycan synthesis and O-linked glycosylation of proteins. For exploring the profiles of glycosylation in the global proteome of Eimeria tenella during develop- mental cycle, and the potential druggable target of EtppGalNAc-T2, we have cloned and recombi- nantly expressed the EtppGalNAc-T2 gene according to its predicted coding sequence deposited in EuPathDB, and evaluated its transcriptional profiling in different stages of life cycle using qRT-PCR method. The results showed that the ORF of EtppGalNAc-T2 is 1 983 bp in full length, and can be expressed in E. coli Transetta （DE3） with recombinant vector EtppGalNAc- T2-pGEX-6p-1. qRT-PCR results showed that the transcriptions of EtppGalNAc-T2 are signifi- cantly varied among different developmental stages, that is, the highest in E. tenella sporozoite, and nearly no-detectable transcription in sporulated occyst. Briefly, these results confirmed O-glycan synthesis process, and may play roles in the regulation of developmental conversion in E. tenella.

关 键 词：柔嫩艾美耳球虫 ppGalNAc-T2 基因克隆 表达差异 QRT-PCR 

分 类 号：S852.723[农业科学—基础兽医学]