十二指肠钩虫锰超氧化物岐化酶基因的克隆与表达

Cloning and expression of the manganese superoxide dismutase (Mn-SOD) gene from Ancylostoma duodenale

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 and express the manganese superoxide dismutase （AdMn-SOD） gene from Ancylostoma duodenale. Methods The full length cDNA from A. duodenale encoding AdMn-SOD was amplified by 3＇RACE and RT-PCR. The nucleotide sequence encoding mature AdMn-SOD was ligated into pET32a to construct the recombinant pET32a/AdMn-SOD plasmid. The recombinant AdMn-SOD was expressed in E. colt BL21 （DE3） by induction with IPTG and was then analyzed by SDS-PAGE. The recombinant protein was affinity-purified with Protino Ni-IDA Resin. Results The AdMn-SOD full-length cDNA in GenBank （accession no. FJ465146） contains an open reading frame of 648 bp, which encodes a protein consisting of 216 amino acids including a signal peptide of 20 amino acid residues. The de- duced AdMn-SOD sequence contained the characteristic motifs of the Mn-SOD family. The mature AdMn-SOD was suc- cessfully ligated into a pET32a plasmid and expressed in E. colt BL21 （DE3） after induction with IPTG. Conclusion Mn-SOD from the human hookworm A. duodenale was cloned and expressed successfully for the first time.