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

Abstract

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.

CLC Number:

S852.723

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

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