十二指肠钩虫天冬氨酸蛋白酶抑制剂的克隆与表达

Cloning and expression of an aspartyl protease inhibitors (Ad-API-1) from Ancylostoma duodenale

目的克隆并表达十二指肠钩虫天冬氨酸蛋白酶抑制剂(Ad-API-1)。方法根据犬钩虫及锡兰钩虫API保守序列设计引物,RT-PCR扩增获得Ad-API-1完整阅读框序列,并将Ad-API-1成熟肽编码序列克隆、连接到原核表达载体pET32a,以构建重组表达质粒,转化至大肠杆菌BL21(DE3)中用IPTG诱导表达,用Ni亲和层析进行纯化。结果成功克隆获得Ad-API-1完整阅读框序列;构建了pET32a/Ad-API-1重组质粒,Ad-API-1在大肠杆菌中得到了高效表达。结论成功克隆并表达Ad-API-1,为进一步研究Ad-API-1的功能,探讨其作为血清学诊断抗原或保护性疫苗奠定了基础。

Objective To clone and express the aspartyl protease inhibitors （Ad-API-1） from Ancylostoma duodenale. Methods The cDNA encoding Ad-API-1 from Ancylostoma duodenale were amplified by RT-PCR. The nucleotide sequence encoding mature Ad-API-1 was ligated into pET32a to construct the recombinant pET32a/Ad-API-1 plasmid. The recombinant Ad-API-1 were expressed in E.coli BL21 （DE3） by inducing with IPTG and purified by Ni-Affinity chromatography. Results The Ad-API-1 gene that deposited in GenBank （accession no. EF490131 ） containing an open reading frame of 687 bp,which encodes a protein consisting of 228 amino acids including a signal peptide of 15 amino acid residues. The mature Ad-API-1 was successfully ligated into pET32a plasmid and expressed in E.coli BL21 （DE3） after inducing by IPTG. Conclusions Ad-API-1 was cloned and expressed successfully for the first time. The recombinant Ad- API-1 may be a potential candidate to be a serodiagnostic reagent or vaccine.