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

doi:10.11843/j.issn.0366-6964.2017.11.019

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (11): 2173-2180.doi: 10.11843/j.issn.0366-6964.2017.11.019

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

刘晓丽^1,2,3, 龚振兴^2,3, 马雪婷^2,3, 曲自刚^2,3, 韩政岚^2,3, 刘保红^2,3, 袁如意^2,3, 杨孝朴^1*, 蔡建平^2,3*

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

收稿日期:2017-05-22 出版日期:2017-11-23 发布日期:2017-11-23
通讯作者: 蔡建平,研究员,博导,主要从事球虫病和猪禽肠道健康研究,E-mail:caijianping@caas.cn;杨孝朴,教授,硕导,主要从事动物生化教学和研究,E-mail:yangxpu@gsau.edu.cn
作者简介:刘晓丽(1988-),女,甘肃会宁人,硕士生,主要从事球虫病研究,E-mail:liuxlil525@163.com
基金资助:
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）

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

LIU Xiao-li^1,2,3, GONG Zhen-xing^2,3, MA Xue-ting^2,3, QU Zi-gang^2,3, HAN Zheng-lan^2,3, LIU Bao-hong^2,3, YUAN Ru-yi^2,3, YANG Xiao-pu^1*, CAI Jian-ping^2,3*

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China;
2. State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
3. Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China

Received:2017-05-22 Online:2017-11-23 Published:2017-11-23

摘要/Abstract

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EtppGalNAc-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EtppGalNAc-T2基因序列设计引物，以RT-PCR方法扩增获得EtppGalNAc-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7 h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EtppGalNAc-T2转录水平的动态变化。结果表明，EtppGalNAc-T2的全长ORF为1 983 bp，编码660个氨基酸，在E.coli Transetta （DE3）可溶性表达。qRT-PCR结果显示，EtppGalNAc-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。

Abstract:

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-acetylgalactosaminyltransferase 2 (ppGalNAc-T2, EC 2.4.1.41) is the first key enzyme in the pathway of O-glycan biosynthesis. Whole genome sequencing of Eimeria 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 developmental cycle, and the potential druggable target of EtppGalNAc-T2, we have cloned and recombinantly 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 significantly 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.

中图分类号:

S852.723

刘晓丽, 龚振兴, 马雪婷, 曲自刚, 韩政岚, 刘保红, 袁如意, 杨孝朴, 蔡建平. 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[J]. 畜牧兽医学报, 2017, 48(11): 2173-2180.

LIU Xiao-li, GONG Zhen-xing, MA Xue-ting, QU Zi-gang, HAN Zheng-lan, LIU Bao-hong, YUAN Ru-yi, YANG Xiao-pu, CAI Jian-ping. Expression Profiling of Polypeptide:N-acetylgalactosaminyltransferase T2 during Eimeria tenella Developmental Cycle[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(11): 2173-2180.

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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

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