十二指肠钩虫ASP-1成熟蛋白基因的克隆、原核表达及纯化

Cloning,expression,and purification of mature ancylostoma-secreted protein 1 of Ancylostoma duodenale

目的获得十二指肠钩虫(Ancylostoma duodenale)分泌蛋白1(mAd-ASP-1)编码基因全长并实现其原核表达。方法根据GenBank AAD13339公布的序列,设计并合成引物,RT-PCR扩增mAd-ASP-1的成熟肽全基因序列,测序后克隆至pET-22b(+)载体,转化大肠埃希菌,诱导表达Ad-ASP-1,亲和层析纯化。结果 RT-PCR扩增获得了mAd-ASP-1基因全长,成功构建了原核表达质粒pET-22b-mAd-ASP-1,转化大肠埃希菌Rosetta-gami 2(DE3)菌株,经IPTG诱导,表达预期47ku的重组蛋白Ad-ASP-1,且主要以可溶形式存在。利用His Trap HP亲和柱获得了纯化的重组蛋白Ad-ASP-1,确定纯化重组蛋白咪唑洗脱液最佳浓度为100mmol/L。Western blot显示,该融合蛋白可被鼠抗His-tag抗体识别。结论获得了mAd-ASP-1基因全长,并成功构建其原核表达体系,为获得大量Ad-ASP-1基因工程产品奠定了实验基础。

Objective To obtain the gene encoding the mature aneylostoma-secreted protein 1 of Ancylostoma duodenale （mAd-ASP-1） and express it in E. coll. Methods In accordance with the published nucleotide sequence of mad ASP 1, primers were designed and synthesized to amply the gene using RT-PCR. After sequencing, the target gene was cloned in-to the pET-22b（＋） plasmid, transformed into E. coli, and purified by affinity chromatography. Results The cDNA coding for mAd-ASP-1 was amplified with RT-PCR, and the plasmid pET 22b-mAd-ASP-1 was successfully constructed. After induction with IPTG, the recombinant protein Ad-ASP-1 was expressed in solution with a relative molecular mass of 47 ku. The recombinant protein Ad-ASP-1 was purified using an affinity column, and the optimal concentration of im-idazole eluate was 100 mmol/L. Western blot analysis revealed that the recombinant mAd-ASP-1 protein could be com bined with mouse anti-His-Tag, so the expressed protein was definitely confirmed to be the target protein. Conclusion The full-length gene coding for mAd-ASP-1 was obtained and a system for its prokaryotic expression was constructed. This provides a basis for large-scale production of the recombinant protein Ad-ASP-1.