关键词: 甜菜夜蛾, 谷氧还蛋白, 原核表达, 融合蛋白, 酶活力

Abstract: 【Aim】 Glutardoxin (Grx) is a thiol-dependent antioxidant enzyme in organisms and plays roles in many important life activities including biological oxidative stress and cell apoptosis. This study aims to analyze the gene sequence of SeGrx1 of the beet armyworm, Spodoptera exigua, and its enzyme-catalyzed reaction characteristics, so as to lay a foundation for revealing the function of SeGrx1 in S. exigua. 【Methods】 Based on the previously obtained transcriptome data of S. exigua, the full-length cDNA of Grx1 gene was cloned by RACE-PCR. The ORF of the gene was linked to the prokaryotic expression vector pET-16b and expressed in Escherichia coli BL21 induced with IPTG. The fusion protein was purified by Ni-NTA chromatographic column and Superdex75/200 molecular sieve. The enzymatic activity and catalytic kinetics of the purified SeGrx1 were detected by fluorescence microplate reader. 【Results】 The full-length cDNA of SeGrx1 (GenBank accession no.: MK318813) of S. exigua is 738 bp in length, with an open reading frame (ORF) of 351 bp flanked by a 5′ non-coding region of 40 bp and a 3′ non-coding region of 347 bp. Bioinformatics and structure analysis showed that SeGrx1 consists of 116 amino acids, with the predicted molecular weight of 12.57 kD. SeGrx1 belongs to dithiol glutaredoxin characterized by including a CPYC active site. SeGrx1 is composed of four parallel and antiparallel β-strands in the center, which are surrounded by five α-helixes. The recombinant plasmid pET-16b-SeGrx1 was successfully constructed and highly expressed in E. coli. The target protein SeGrx1 with the purity of more than 95% was obtained by optimizing the induction and purification conditions. With hGrx1 of human as the standard, the specific activity of the purified SeGrx1 was 0.577 U/mg pro, the V_max value was 20.5 U/mg pro, and the Km value was 14.3 nmol/L. 【Conclusion】 In this study SeGrx1 of S. exigua was successfully expressed in vitro, and its enzymatic kinetic parameters were obtained, providing a foundation for further exploring the biological function of this protein and its application in pest control.

Key words: Spodoptera exigua; glutaredoxin, prokaryotic expression, fusion protein, enzyme activity