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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 clone and express the gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Methods GAPDH gapC gene fragment was amplified from the genome of Erysipelas virbatus C43065 by PCR and cloned into the BamHⅠ and KpnⅠ cloning sites of prokaryotic expression vector pET32a (+) to construct the recombinant plasmid pET32a-gapC. Escherichia coli BL21 (DE3) was induced by IPTG to express the recombinant protein GapC with Trx at its N-terminus. The expressed product was detected by SDS-PAGE and Western blot. Results The amplified fragment of gapC gene of C43065 strain was 1 005 bp in length, which was 99% homologous to the nucleotide sequence of the gapC gene of the reported strain of Erysipengia Fujisawa. SDS-PAGE results showed that the expressed protein molecular mass unit is about 57ku, which is similar to the expected size. Western blot results showed that the expressed recombinant protein GapC specifically reacted with anti-6 histidine tag mouse monoclonal antibody. Conclusion The successful expression of GapC protein in Escherichia coli expression system can be used to further study the biological function of GapC.