柔嫩艾美耳球虫5-磷酸脱氧木酮糖还原异构酶基因的克隆、原核表达及酶活性分析

doi:10.11843/j.issn.0366-6964.2015.04.017

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (4): 631-636.doi: 10.11843/j.issn.0366-6964.2015.04.017

柔嫩艾美耳球虫5-磷酸脱氧木酮糖还原异构酶基因的克隆、原核表达及酶活性分析

廖申权，吴彩艳，戚南山，李娟，吕敏娜，张健騑，谢明权，孙铭飞^*

（广东省农业科学院动物卫生研究所，广东省畜禽疫病防治研究重点实验室，广东省兽医公共卫生公共实验室，广州 510640）

收稿日期:2014-07-28 出版日期:2015-04-23 发布日期:2015-04-23
通讯作者: 孙铭飞，副研究员，E-mail：smf7810@126.com
作者简介:廖申权（1981-），女，重庆荣昌人，博士，主要从事鸡球虫生化代谢研究，E-mail：lsq6969@163.com
基金资助:
国家自然科学基金项目 (31201902；31302087；31402186)；广州市珠江科技新星项目（2012J2200059）；广东省自然科学基金项目（S2013040015220）；广东省科技计划项目（2011B050700007）；广州市科技计划项目（2014J4100230）；广东省农业科学院院长基金（201413）

Cloning，Prokaryotic Expression and Enzymatic Activity of 1-deoxy-D-xylulose-5-phosphate Reductoisomerase of E.tenella

LIAO Shen-quan，WU Cai-yan，QI Nan-shan，LI Juan，LÜ Min-na，ZHANG Jian-fei，XIE Ming-quan，SUN Ming-fei^*

(Guangdong Laboratory for Animal Diseases,Guangdong Open Laboratory of Veterinary Public Health,Institute of Animal Health，Guangdong Academy of Agricultural Sciences，Guangzhou 510640，China)

Received:2014-07-28 Online:2015-04-23 Published:2015-04-23

摘要/Abstract

摘要：

旨在克隆柔嫩艾美耳球虫5-磷酸脱氧木酮糖还原异构酶（EtDXR）基因，初步分析EtDXR的酶活性。根据EuPathDB数据库信息注释、拼接Etdxr基因序列，设计特异性引物扩增Etdxr基因，构建原核表达质粒pCold I-EtDXR，转化E.coli Rosetta（DE3），IPTG诱导表达，进行SDS-PAGE及Western blot分析，纯化rEtDXR，对rEtDXR进行酶活性分析，测定pH和Mg²⁺离子浓度对酶活性的影响。结果显示，Etdxr基因完整编码序列为1 746 bp，氨基酸序列具有与其他物种高度保守的酶活性中心；成功构建原核表达质粒pCold I-EtDXR，SDS-PAGE以及Western blot分析显示，得到约50 ku的蛋白质；利用直接检测底物NADPH 消耗速率的方法分析EtDXR酶活性，结果显示，不同pH和离子浓度对EtDXR的酶活性有显著影响，其最佳的酶反应条件为pH 7.0，Mg²⁺离子浓度4.5 mmol•L^-1。本研究为进一步以EtDXR为药靶筛选抗球虫先导化合物奠定了基础。

Abstract:

The aim of this study was to clone 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene in Eimeria tenella，and analyze its enzymatic activity.The predicted sequences of Etdxr were obtained from EuPathDB by bioinformatics analysis.The full-length cDNA was amplified by PCR and then cloned into pCold I vector.The recombinant plasmid was transformed into E.coli Rosetta (DE3) and induced by IPTG.The expression products were analyzed by SDS-PAGE and Western blot，and then were purified.We further evaluated the enzymatic activity of EtDXR by monitoring the consumption of NADPH.The results showed that the open reading frame of E.tenella DXR was 1 746 bp.Further analysis of the amino acid sequence revealed that EtDXR contains the conserved domains with other DXRs.The results showed that the recombinant vector pCold I-EtDXR was constructed successfully.The SDS-PAGE and Western blot results showed that the purified protein was 50 kDa.The characterizations of reaction conditions used in the experiments，such as pH and ion concentration have significant effects on the enzymatic activity of rEtDXR.According to the response surface plots，the maximum enzymatic activity was pH 7.0 and 4.5 mmol•L^-1 Mg²⁺.This study provides a foundation for the further study to select inhibitors of E.tenella DXR.

中图分类号:

S852.723

廖申权，吴彩艳，戚南山，李娟，吕敏娜，张健騑，谢明权，孙铭飞. 柔嫩艾美耳球虫5-磷酸脱氧木酮糖还原异构酶基因的克隆、原核表达及酶活性分析[J]. 畜牧兽医学报, 2015, 46(4): 631-636.

LIAO Shen-quan，WU Cai-yan，QI Nan-shan，LI Juan，Lü Min-na，ZHANG Jian-fei，XIE Ming-quan，SUN Ming-fei. Cloning，Prokaryotic Expression and Enzymatic Activity of 1-deoxy-D-xylulose-5-phosphate Reductoisomerase of E.tenella[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(4): 631-636.

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柔嫩艾美耳球虫5-磷酸脱氧木酮糖还原异构酶基因的克隆、原核表达及酶活性分析

Cloning，Prokaryotic Expression and Enzymatic Activity of 1-deoxy-D-xylulose-5-phosphate Reductoisomerase of E.tenella

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