摘要
目的克隆和表达红斑丹毒丝菌甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因。方法采用PCR从红斑丹毒丝菌C43065株基因组中扩增编码GAPDH的gapC基因片段,将其克隆至原核表达载体pET32a(+)的BamHⅠ和KpnⅠ多克隆位点上,构建重组载体pET32a-gapC,转化大肠埃希菌BL21(DE3),在IPTG诱导下表达N端带有Trx的重组蛋白GapC,用SDS-PAGE和Western blot检测表达产物。结果扩增得到的C43065株gapC基因片段大小为1 005bp,与已报道的红斑丹毒丝菌Fujisawa株gapC基因核苷酸序列的同源性为99%。SDS-PAGE结果显示表达蛋白分子质量单位约为57ku,与预期的大小相近。Western blot检测结果显示表达的重组蛋白GapC能与抗6个组氨酸标签鼠单克隆抗体发生特异性反应。结论用大肠埃希菌表达系统成功表达GapC蛋白,可用于红斑丹毒丝菌GapC生物学功能的进一步研究。
Objective To express the glyceraldehyde-3-phosphate dehydrogenase（GAPDH）gene of Erysipelothrix rhusiopathiae in Escherichia coli. Methods The gapC gene encoding the GAPDH gene was amplified from the genomic DNA of E.rhusiopathiae C43065 with PCR and that gene was then cloned into the prokaryotic expression vector pET32a（＋）to yield the recombinant plasmid pET32a-gapC.In E.coli BL21（DE3）harboring the recombinant expression plasmid pET32a-gapC,expression of the fusion protein Trx-GapC was induced with IPTG.The expressed protein was identified with SDS-PAGE and Western blotting. Results The open reading frame of the gapC gene was 1005 bp in length,and the nucleotide sequence of the gapC gene from the C43065 strain was 99%similar to that of the gapC gene in the previously reported Fujisawa strain of E.rhusiopathiae.SDS-PAGE analysis revealed a single protein band with a molecular weight of 57 ku,and Western blotting indicated that the Trx-GapC fusion protein was recognized specifically by anti-HisTag mouse monoclonal antibodies. Conclusion A Trx-GapC fusion protein of E.Rhusiopathiae was successfully expressed in E.coli BL21 via a prokaryotic expression vector.The recombinant protein can be used for further study of the biological function of GapC in E.rhusiopathiae.
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第1期5-8,12,共5页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.31360613
31072142)
关键词
红斑丹毒丝菌
甘油醛-3-磷酸脱氢酶
基因克隆
原核表达
Erysipelothrix rhusiopathiae
glyceraldehyde-3-phosphate dehydrogenase
gene cloning
prokaryotic expression
