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目的克隆十二指肠钩虫锰超氧化物岐化酶(AdMn-SOD)基因,并在大肠埃希菌中表达。方法用3′RACE及RT-PCR技术扩增获得AdMn-SOD全长cDNA编码序列;设计引物,克隆AdMn-SOD成熟肽编码序列,连接到原核表达载体pET32a,构建重组表达质粒,转化大肠埃希菌BL21(DE3)并用IPTG诱导表达,SDS-PAGE分析表达情况,诱导表达的重组蛋白用Ni亲和层析进行纯化。结果成功克隆获得AdMn-SOD全长cDNA序列,推导编码的氨基酸序列具有Mn-SOD的保守结构特征;构建了pET32a/AdMn-SOD原核表达重组质粒,AdMn-SOD在大肠埃希菌中得到高效表达,表达的融合蛋白的分子质量单位约为40ku。结论成功克隆并表达了十二指肠钩虫锰超氧化物岐化酶基因,为进一步了解十二指肠钩虫锰超氧化物岐化酶的特性与功能奠定了基础。
Objective To clone the gene of manganese superoxide dismutase (AdMn-SOD) of hookworm, and express it in Escherichia coli. Methods The full-length cDNA encoding sequence of AdMn-SOD was amplified by 3’RACE and RT-PCR. Primer was designed to clone the mature peptide sequence of AdMn-SOD and ligated into the prokaryotic expression vector pET32a to construct a recombinant plasmid. The recombinant plasmid was transformed into E. coli The strain BL21 (DE3) was induced with IPTG, and the expression was analyzed by SDS-PAGE. The recombinant protein induced by IPTG was purified by Ni affinity chromatography. Results The full-length cDNA sequence of AdMn-SOD was successfully cloned, and the deduced amino acid sequence of the recombinant protein possessed the conserved structural features of Mn-SOD. The prokaryotic expression recombinant plasmid pET32a / AdMn-SOD was constructed and the recombinant AdMn-SOD was highly expressed in Escherichia coli , The expressed fusion protein molecular mass unit is about 40ku. Conclusion The Mn-SOD gene was successfully cloned and expressed, which lays the foundation for further understanding of the characteristics and functions of manganese superoxide dismutase of H. duodenum.