CAJ | 학술논문

为研究柔嫩艾美耳球虫（Eimeria tenella）葡萄糖-6-磷酸异构酶（glucose-6-phosphate isomerase, G6-PI）的生化特性，本研究对其基因进行了克隆和原核表达。根据NCBI已公布的E. tenella Houghton株G6-PI的基因序列（GI：298161921）设计特异性引物，运用RT-PCR方法扩增EtG6-PI基因序列，并将其克隆到表达载体pColdI、pCold43a中，构建重组质粒pColdI-EtG6-PI、pCold43a-EtG6-PI，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， E. tenella G6-PI的ORF序列（1650 bp），编码550个氨基酸；重组菌pColdI-EtG6-PI、pCold43a-EtG6-PI均能成功诱导表达，其中pCold43a-EtG6-PI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G6-PI基因的成功克隆表达为该酶的功能研究奠定了基础。
위연구유눈애미이구충（Eimeria tenella）포도당-6-린산이구매（glucose-6-phosphate isomerase, G6-PI）적생화특성，본연구대기기인진행료극륭화원핵표체。근거NCBI이공포적E. tenella Houghton주G6-PI적기인서렬（GI：298161921）설계특이성인물，운용RT-PCR방법확증EtG6-PI기인서렬，병장기극륭도표체재체pColdI、pCold43a중，구건중조질립pColdI-EtG6-PI、pCold43a-EtG6-PI，경측서감정정학후，장기전화감수태세포E.coli Rosetta (DE3)，병진행IPTG유도표체。결과표명， E. tenella G6-PI적ORF서렬（1650 bp），편마550개안기산；중조균pColdI-EtG6-PI、pCold43a-EtG6-PI균능성공유도표체，기중pCold43a-EtG6-PI표체부분가용성단백，표체산물순화후가용우매생화특성분석。유눈애미이구충G6-PI기인적성공극륭표체위해매적공능연구전정료기출。
In order to analyze biochemical properties of Eimeria tenella glucose-6- phosphate isomerase EtG6-PI, the full-length of open reading frame Etg6-PI was amplified in RT-PCR from total RNA from the second-generation schizonts of E.tenella. The amplified fragments were Results showed that the open reading frame was 1650 bp, sequenced. The Etg6-PI gene was cloned into the expression vectors pColdI and pCold43a and expressed in E. coli Rosetta (DE3). encoded a protein of 550 amino acids. The recombinant vectors pColdI-EtG6-PI or pCold43a-EtG6-PI was transformed into E. coli Rosetta (DE3) cells and the expression was induced with IPTG Soluble protein obtained from pCold43a-EtG6-PI purifide by the affinity chromatography assay can be used for biochemical analysis of this enzyme. The prokaryotic expression of E.tenella G6-PI set important basis for function study on glucose-6-phosphate isomerase.