摘要： 亚硝基谷胱甘肽还原酶（S-nitrosoglutathione reductase，GSNOR）在生物体中调控信号分子一氧化氮（NO）的代谢和平衡。为了深入研究GSNOR的功能，利用RT-PCR和RACE （Rapid-amplification of cDNA ends）技术从菠菜根中克隆了SoGSNOR基因的全长序列，该基因编码框1 140 bp，编码379个氨基酸，GenBank登录号为KR381778。将其克隆到原核表达载体pET32a上，构建原核表达载体pET32a-SoGSNOR，并转化大肠杆菌BL21 star （DE3），通过IPTG诱导重组SoGSNOR在大肠杆菌BL21 star （DE3）中高效表达，表达的融合蛋白分子质量约为65 ku，且在上清液和包涵体中均有表达，可溶性部分经Ni²⁺ NTA亲和柱纯化，获得纯化蛋白，并注射昆明小鼠，制备多克隆抗体，并对其进行了Western Blot检测，结果表明，稀释10 000倍的抗血清能够检测到与预期SoGSNOR大小一致的目的条带。试验结果为进一步研究菠菜SoGSNOR基因功能奠定了基础。

关键词: S-亚硝基谷胱甘肽还原酶, 原核表达, 菠菜

Abstract: S-nitrosoglutathione reductase (GSNOR) can modulate metabolism and homeostasis of NO in organisms. In order to further study GSNOR function, the full-length cDNA encoding SoGSNOR was amplified by RT-PCR and RACE (Rapid-amplification of cDNA ends).The open reading frame of SoGSNOR was 1 140 bp,encoding 379 amino acids (GenBank accession No.KR381778).The SoGSNOR was inserted into pET32a.The correct recombinant pET32a-SoGSNOR was transformed into E.coli BL21 star (DE3).The SoGSNOR-His fusion protein was induced by isopropyl-beta-D-thiogalactoside (IPTG).SDS-PAGE analysis showed that the recombinant protein with a molecular mass about 65 ku was highly expressed in E.coli BL21 star(DE3) and existed both in the supernatant and the precipitation part of E.coli lysates.The supernatant was further purified by Ni²⁺ NTA affinity chromatography and immunized white mouse as an antigen.The polyclonal antibody was obtained and analyzed by Western Blot and a specific band was detected which was corresponding to the expected size of SoGSNOR.The study laid a foundation for further investigating the function of SoGSNOR.

Key words: S-nitrosoglutathione reductase, Prokaryotic expression, Spinach

中图分类号: 

• Q78

• S636.03