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柔嫩艾美耳球虫Cytbc1基因的克隆、序列分析及重组表达 被引量:1

Cloning,sequence analysis and prokaryotic expression of cytochrome bc1 of Eimeria tenella
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摘要 为研究柔嫩艾美耳球虫(Eimeria tenella)细胞色素bc 1复合物(cytochrome bc1,Cytbc1)的生化特性,本研究对其基因进行了克隆和原核表达。根据EupathDB已公布的E.tenella Houghton株Cytbc1基因序列(Contig ID:ETH 00007855)设计特异性引物,运用RT-PCR方法扩增EtCytbc1基因序列,并将其克隆到表达载体pCold43a中,构建重组质粒pCold43a-EtCytbc1,经测序鉴定正确后,将其转化感受态细胞E.coli Rosetta(DE3),并进行IPTG诱导表达。结果表明,EtCytbc1的ORF序列(687bp),编码228个氨基酸;重组菌pCold43a-EtCytbc1能成功诱导表达,pCold43a表达部分可溶性蛋白,表达产物纯化后可用于酶生化特性分析。柔嫩艾美耳球虫Cytbc1基因的成功克隆表达为该酶的功能研究奠定了基础。 In order to analyze biochemical properties of E.tenella cytochrome bc1(EtCytbc1),the full-length of open reading frame of cytochrome bc1 was amplified by RT-PCR from total RNA from the second-generation schizonts of E.tenella.And then cloned into pCold43 avector.This plasmid was transformed into E.coli Rosetta(DE3)and induced by IPTG.The expression products were analyzed by SDS-PAGE and then purified.The results showed that the Cytbc1 gene consists of a 687 bp open reading frame encoding 228 amino acids.Sequencing analysis showed that the recombinant vector pCold43a-EtCytbc1 was constructed successfully.The SDS-PAGE results showed that the recombinant proteins were partially soluble.Soluble protein was obtained from pCold43aEtCytbc1,and was purifide by the affinity chromatography assay.The purified protein can be used for biochemical analysis of this enzyme.The prokaryotic expression of EtCytbc1 provides a basis on the molecular characterization of E.tenella cytochrome bc1.
作者 张晨旭 廖申权 戚南山 吴彩艳 李娟 吕敏娜 林栩慧 吴玄光 孙铭飞 ZHANG Chen-xu ,LIAO Shen-quan ,QI Nan-shan,WU Cai-yan ,LI Juan ,LYU Min-na ,LIN Xu- hui , WU Xuan-guang , SUN Ming-fei (1. College of Veterinary Medicine, South China Ag- ricultural university ,Ouangzhou 510640,China;2. Institute for Animal Health ,Ouangdong Acad- emy Agricultural Sciences ,Guangzhou 510640,China)
出处 《中国兽医学报》 CAS CSCD 北大核心 2016年第3期448-453,共6页 Chinese Journal of Veterinary Science
基金 国家自然科学基金资助项目(31201902 31302087) 珠江科技新星资助项目(2012J2200059) 广东省自然科学基金资助项目(S2013040015220) 广东省农业科学院院长基金资助项目(201413)
关键词 柔嫩艾美耳球虫 细胞色素bc1复合物 克隆 原核表达 Eimeriatenella cytochrome bc1 cloning prokaryotic expression
作者简介 张晨旭(1989),女,硕士。 通讯作者,E-mail:wuxg62@scau.edu.cn; 通讯作者,E-mail:smf7810@gmail.com
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