重组人SHH蛋白N端在HEK293T细胞中的分泌表达与鉴定

Secretory Expression and Identification of Recombinant Human Sonic Hedgehog Protein N-Terminal Domain by HEK293T Cells

目的:获得有活性的Sonic Hedgehog(SHH)蛋白N端结构域蛋白纯品,该结构域是SHH蛋白与受体结合结构域,可用作抗原,用于研制抗SHH的中和抗体。方法:应用PCR技术从商业化人Shh基因中分别扩增该基因5'端591和600 bp的片段,并插入真核表达载体pL293,分别在HEK293T细胞中进行瞬时分泌表达,通过His标签纯化后获得SHH-591和SHH-600蛋白纯品,SDS-PAGE和Western印迹对表达产物进行分析,并通过ELISA进行结合活性鉴定。结果:构建了重组表达载体pL293-Shh-N591和pL293-Shh-N600,酶切鉴定和测序证实含有目的基因片段,真核瞬时表达产物均在相对分子质量约20×103处可见与预期相符的条带,该条带可被His标签抗体所识别;纯化获得了SHH-591-His和SHH-600-His蛋白纯品;ELISA结合实验结果显示SHH-591-His和SHH-600-His均能与抗His标签抗体结合,而SHH-591-His与SHH中和抗体的结合能力更强。结论:获得了真核表达的SHH-N蛋白SHH-591-His,可用于下一步中和抗体药物的筛选和后续研究。

Objective： To obtain active recombinant sonic hedgehog（SHH） protein N-terminal domain, which is the binding domain to SHH receptor, and it could be used as antigen for preparing neutralizing antibody against SHH. Methods： The aim genes were prepared from the cDNA of human Shh gene obtained from market by PCR, and were cloned into expressing vector pL293 for eukaryotic expression. The recombinant protein named SHH-591 and SHH-600 were expressed and secreted into the culture supernatant, and the purified proteins were obtained by His-tag affinity purification, after which, the proteins were analyzed by SDS-PAGE and Western bolt, as well as ELISA for binding activity. Results： The recombinant eukaryotic expression vector pL293-Shh-N591 and pL293- Shh-N600 were constructed and confirmed by restriction endonuclease analysis and sequencing. The purified recombinant proteins of SHH-591-His and SHH-600-His were obtained. The result of SDS-PAGE showed a specific lane at 20 kDa for both, which could be identified by His-tag antibody. Although two proteins could bind Histag antibody, but only SHH-591-His could bind an neutralizing antibody of SHH strongly. Conclusion： Recombinant SHH-N named SHH- 591- His were obtained using eukaryotic expression and identified, which laid a good foundation for further study and preparing the therapeutic antibody against SHH.