柔嫩艾美耳球虫真核起始因子5和5A基因的克隆表达及转录动态分析

doi:10.11843/j.issn.0366-6964.2014.12.016

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (12): 2019-2027.doi: 10.11843/j.issn.0366-6964.2014.12.016

柔嫩艾美耳球虫真核起始因子5和5A基因的克隆表达及转录动态分析

苟灵俏^1，2，龚振兴²，于三科¹，殷昊²，马雪婷²，蔡建平^2*，林青^1*

（1.西北农林科技大学动物医学院，杨凌 712100；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）

收稿日期:2014-04-21 出版日期:2014-12-23 发布日期:2014-12-23
通讯作者: 蔡建平，E-mail：caijianping@caas.cn；林青，E-mail：yllinqing@126.com
作者简介:苟灵俏（1988-），女，河南周口人，硕士生，主要从事动物疫病防治研究， E-mail：goulingqiao1120@126.com
基金资助:
中央高校基本科研业务费专项资金（QN2012018）；国家自然科学基金（31272554）

Cloning and Expression Profiling of Eukaryotic Initiation Factor 5 and 5A Genes in Eimeria tenella

GOU Ling-qiao^1,2，GONG Zhen-xing²，YU San-ke¹，YIN Hao²，MA Xue-ting²，CAI Jian-ping^2*，LIN Qing^1*

(1.College of Veterinary Medicine，Northwest A&F University，Yangling 712100，China；2.National Key Laboratory of Veterinary Etiological Biology，Gansu Province Key Laboratory of Animal Parasitic Diseases，Lanzhou Veterinary Research Institute，Chinese Academy of Agricultural Sciences，Lanzhou 730046，China)

Received:2014-04-21 Online:2014-12-23 Published:2014-12-23

摘要/Abstract

摘要：

旨在研究柔嫩艾美耳球虫（E.tenella）真核起始因子5（EteIF5）和5A（EteIF5A）的功能，克隆、表达并分析EteIF5和EteIF5A在E.tenella广东株不同发育阶段的表达量变化。根据E.tenella基因组数据预测的编码序列设计引物，采用RT-PCR方法克隆EteIF5和EteIF5A的ORF，插入酶切位点后连接到原核表达载体pMAL-c2x进行诱导表达。提取E.tenella 5个发育阶段（/时期）的总RNA，使用qRT-PCR检测EteIF5和EteIF5A转录的动态变化。测序结果表明，克隆获得的EteIF5和EteIF5A ORF全长分别为1 248和486 bp，各自编码315和161个氨基酸，均在大肠埃希菌表达系统中成功表达。qRT-PCR定量分析显示，eIF5和eIF5A的mRNA在E.tenella不同发育阶段转录量不同，子孢子阶段最高，在孢子化卵囊阶段最低。首次克隆获得了E.tenella eIF5和eIF5A ORF，揭示EteIF5和EteIF5A mRNA的转录量在不同发育阶段有显著差异。试验结果为进一步研究EteIF5和EteIF5A的功能，从EteIF5A合成途径研制新型抗球虫药提供了试验基础。

关键词: 柔嫩艾美耳球虫, 真核起始因子, 基因克隆, 表达差异, qRT-PCR

Abstract:

In order to study the functions of eukaryotic initiation factors (eIFs) of Eimeria tenella，the genes encoding eIF5 (EteIF5) and eIF5A (EteIF5A) were cloned and detected for evaluating their transcriptional profiling during E.tenella different developmental stages.The primers were designed based on the predicted ORF sequences from E.tenella (Houghton) whole genome data （http://www.eupathdb.org），and the ORFs of EteIF5 and EteIF5A were cloned from Guangdong strain of E.tenella sporulated oocyst total RNA by RT-PCR.The ORF sequences of EteIF5 and EteIF5A were inserted into the prokaryotic expression vector pMAL-c2x，and expressed in Escherichia coli，respectively.Five total RNA samples were extracted from 5 developmental stages of E.tenella，i.e.，unsporulated oocysts，sporulating for 7 hours，sporulated oocysts，sporozoites，and 2^ndgeneration merozoites，and subsequently were transcripted into the first strand of cDNA.The E.tenella β-actin gene was chosen as a control，and the transcriptional levels of EteIF5 and EteIF5A were detected by Real-time RT-PCR method.The sequencing results showed that cloned ORF sequences of EteIF5 and EteIF5A are 1 248 bp and 486 bp in their length，encoding the peptides composed of 315 and 161 amino acids，respectively.The qRT-PCR results revealed that EteIF5 and EteIF5A showed different transcription levels through the E.tenella developmental stages，and both of EteIF5 and EteIF5A in sporozoites were all higher than other stages.The cloning and expression profiling of EteIF5 and EteIF5A genes of E.tenella was studied firstly as we known，and these results will provide an important basis for probing the functions of E.tenella eIFs and be helpful to provide more insights into development of novel anti-coccidials.

Key words: Eimeria tenella, eIFs, gene cloning, expression profile, qRT-PCR

中图分类号:

S855.9

苟灵俏，龚振兴，于三科，殷昊，马雪婷，蔡建平，林青. 柔嫩艾美耳球虫真核起始因子5和5A基因的克隆表达及转录动态分析[J]. 畜牧兽医学报, 2014, 45(12): 2019-2027.

GOU Ling-qiao，GONG Zhen-xing，YU San-ke，YIN Hao，MA Xue-ting，CAI Jian-ping，LIN Qing. Cloning and Expression Profiling of Eukaryotic Initiation Factor 5 and 5A Genes in Eimeria tenella[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(12): 2019-2027.

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柔嫩艾美耳球虫真核起始因子5和5A基因的克隆表达及转录动态分析

Cloning and Expression Profiling of Eukaryotic Initiation Factor 5 and 5A Genes in Eimeria tenella

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