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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 duodenal hookworm aspartic protease inhibitor (Ad-API-1). Methods According to the canine hookworm and hookworm Cereal API API, primers were designed and the complete reading frame of Ad-API-1 was amplified by RT-PCR. The mature coding sequence of Ad-API-1 was cloned and linked to the prokaryotic expression vector pET32a, To construct a recombinant expression plasmid, which was transformed into E. coli BL21 (DE3) for expression with IPTG and purified by Ni affinity chromatography. Results The complete reading frame of Ad-API-1 was successfully cloned. Recombinant plasmid pET32a / Ad-API-1 was constructed and highly expressed in Escherichia coli. Conclusion The successful cloning and expression of Ad-API-1 laid the foundation for the further study on the function of Ad-API-1 and its application as a serological diagnostic or protective vaccine.