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.

Key words: Eimeria tenella, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, cloning, prokaryotic expression, enzymatic activity

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.

References

［1］SATO S.The apicomplexan plastid and its evolution ［J］.Cell Mol Life Sci，2011，68(8):1285-1296.
［2］GOBLE J L，ADENDORFF M R，DE BEER T A，et al.The malarial drug target Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR)：development of a 3-D model for identification of novel，structural and functional features and for inhibitor screening ［J］.Protein Pept Lett，2010，17(1):109-120.
［3］BORRMANN S，LUNDGREN I，OYAKHIROME S，et al.Fosmidomycin plus clindamycin for treatment of pediatric patients aged 1 to 14 years with Plasmodium falciparum malaria ［J］.Antimicrob Agents Chemother，2006，50(8):2713-2718.
［4］XIE M Q，GILBERT J M，FULLER A L，et al.A new method for purification of Eimeria tenella merozoites ［J］. Parasitol Res，1990，76(7)：566-569.
［5］SUN M，ZHU G，QIN Z，et al.Functional characterizations of malonyl-CoA:acyl carrier protein transacylase (MCAT) in Eimeria tenella ［J］.Mol Biochem Parasitol，2012，184（1）：20-28.
［6］DHIMAN R K，SCHAEFFER M L，BAILEY A M，et al.1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) from Mycobacterium tuberculosis：towards understanding mycobacterial resistance to fosmidomycin ［J］. J Bacteriol，2005，187(24):8395-8402.
［7］SEEBER F，SOLDATI-FAVRE D.Metabolic pathways in the apicoplast of apicomplexa ［J］.Int Rev Cell Mol Biol，2010，281：161-228.
［8］JOMAA H，WIESNER J，SANDERBRAND S，et al.Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs ［J］. Science，1999，285(5433):1573-1576.
［9］ROOS D S.The apicoplast as a potential therapeutic target in Toxoplasma and other apicomplexan parasites：some additional thoughts ［J］.Parasitol Today，1999，15(1):41.
［10］DE RUYCK J，WOUTERS J，POULTER C D.Inhibition studies on enzymes involved in isoprenoid biosynthesis：Focus on two potential drug targets：DXR and IDI-2 enzymes ［J］.Curr Enzym Inhib，2011，7(2)：79-95.
［11］BEHRENDT C T，KUNFERMANN A，ILLARIONOVA V，et al.Reverse fosmidomycin derivatives against the antimalarial drug target IspC (Dxr) ［J］.J Med Chem，2011，54(19):6796-6802.
［12］YADAVA A，OCKENHOUSE C F.Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems ［J］.Infect Immun，2003，71(9):4961-4969.
［13］CHELESKI J，FREITAS R F，WIGGERS H J，et al.Expression，purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi ［J］.Protein Expres Purif，2011，76:190-196.
［14］UMEDA T，TANAKA N，KUSAKABE Y，et al.Molecular basis of fosmidomycin′s action on the human malaria parasite Plasmodium falciparum ［J］. Sci Rep，2011，1:9.
［15］JACKSON E R，DOWD C S.Inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr)：a review of the synthesis and biological evaluation of recent inhibitors ［J］.Curr Top Med Chem，2012，12(7):706-728.

[1]	OUYANG Qingyuan, WANG Yushi, HU Shenqiang, LI Liang, LIU Hehe, HE Hua, WANG Jiwen. Cloning of GATA4/5/6 Genes and Their Expression Patterns in Duck Hearts and Livers during Postnatal Development [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1775-1786.
[2]	GAO Junying, ZHANG Dongchao, LI Xuan, WANG Hualin, JIANG Ning. The Characteristic Study of Toxoplasma gondii Surface Antigen SAG1 Adhering to Heparan Sulfate on the Host Cell Surface [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(9): 1874-1881.
[3]	WANG Junwei, CHEN Fangzhou, KU Xugang, LI Chang, HE Qigai. Establishment and Clinical Application of an Indirect ELISA for Detection of Antibodies against PCV3 Based on Recombinant ORF2 Protein [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(2): 454-460.
[4]	WANG Junwei, CHEN Fangzhou, KU Xugang, LI Chang, HE Qigai. Establishment and Clinical Application of an Indirect ELISA for Detection of Antibodies against PCV3 Based on Recombinant ORF2 Protein [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(2): 454-460.
[5]	CHEN Rui, ZHU Zifeng, HUANG Jian, YANG Xiaonong. Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 61-70.
[6]	WANG Yanfang, ZHOU Yaping, GUO Ting, ZHANG Xinzhu, WU Qian, CHEN Xindi, HAO Yongqing. Cloning, Expression, Subcellular Localization and Indirect ELISA Method of EF-Tu Gene in Mycoplasma bovis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 134-142.
[7]	CHEN Rui, ZHU Zifeng, HUANG Jian, YANG Xiaonong. Molecular Cloning and Expression Analysis of Short Stature Homeobox 2 (Shox2) in Oryctolagus cuniculus [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 61-70.
[8]	WANG Yanfang, ZHOU Yaping, GUO Ting, ZHANG Xinzhu, WU Qian, CHEN Xindi, HAO Yongqing. Cloning, Expression, Subcellular Localization and Indirect ELISA Method of EF-Tu Gene in Mycoplasma bovis [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 134-142.
[9]	ZHANG Yuan-yuan, XU Rong, HE Chuan-chuan, LI Xiao-chun, CHENG Gui-feng, GUO Lu, LIU Jin-ming, LI Hao, GU Shao-peng, JIN Ya-mei. Cloning,Expression and Functional Analysis of ELAV-like 2 Gene of Schistosoma japonicum [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1987-1997.
[10]	ZHANG Yuan-yuan, XU Rong, HE Chuan-chuan, LI Xiao-chun, CHENG Gui-feng, GUO Lu, LIU Jin-ming, LI Hao, GU Shao-peng, JIN Ya-mei. Cloning,Expression and Functional Analysis of ELAV-like 2 Gene of Schistosoma japonicum [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(9): 1987-1997.
[11]	FENG Yan, GUO Jia, XU Rui-qin, MA Si-xu, WEI Feng-ling, ZHANG Liu-jun, YANG Guo-yu, XIA Ping-an, ZHANG Gai-ping. Comparison of Neutralizing Antibodies Induced by PRRSV GP5 Recombinant Proteins in Different Forms of Truncation [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1222-1230.
[12]	LI Cheng, ZHANG Yu-di, HUANG Xiao-bo, LIU Hao-yu, LIU Zhi-peng, ZHAO Yu-jia, CAO San-jie, WEN Xin-tian, WEN Yi-ping, ZHAO Qin, WU Rui. Cloning and Prokaryotic Expression of Porcine Deltacoronavirus S1 Gene and Preparation of Polyclonal Antibody [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1256-1264.
[13]	LI Cheng, ZHANG Yu-di, HUANG Xiao-bo, LIU Hao-yu, LIU Zhi-peng, ZHAO Yu-jia, CAO San-jie, WEN Xin-tian, WEN Yi-ping, ZHAO Qin, WU Rui. Cloning and Prokaryotic Expression of Porcine Deltacoronavirus S1 Gene and Preparation of Polyclonal Antibody [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1256-1264.
[14]	FENG Yan, GUO Jia, XU Rui-qin, MA Si-xu, WEI Feng-ling, ZHANG Liu-jun, YANG Guo-yu, XIA Ping-an, ZHANG Gai-ping. Comparison of Neutralizing Antibodies Induced by PRRSV GP5 Recombinant Proteins in Different Forms of Truncation [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1222-1230.
[15]	LI Zhi-peng, LIU Fu-hang, CUI Kui-qing, SHI De-shun, LIU Qing-you. Prokaryotic Expression and Purification of Ferritin and Nano-particles Self-assembling in vitro [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 75-82.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments