基因重组型人SORL1蛋白质片段的原核表达和纯化

目的制备基因重组型人SORL1蛋白质片段,为探讨阿尔茨海默病的发病机制提供实验基础。方法采用PCR方法扩增人sorl1 cDNA编码区5 923～6 260 bp片段,克隆入原核表达载体pET-28a(+)中。用大肠杆菌BL21(DE3)plysS表达重组质粒,用液相色谱法纯化基因重组蛋白。结果 PCR扩增产物为358 bp,其ATG和TGA之间的结构与人sorl1 cDNA的5 923～6 260 bp区完全一致。基因重组质粒在大肠杆菌BL21(DE3)plysS中高效表达,其表达产物存在于包涵体组分中。纯化的基因重组蛋白在SDS-PAGE中表现为单一条带,表观分子量约为13 000。结论人sorl1 cDNA的5 923～6 260 bp片段在原核细胞中大量表达,纯化后的基因重组蛋白可尝试用于抗体的制备。

Prokaryotic expression and purification of recombinant human SORL1 protein

Objective To prepare the recombinant human SORL1 protein, and provide the experimental foundation for study about the pathogenesis of Alzheimer's disease. Methods A human sorl1 cDNA fragment(5 923~6 260 bp) was amplified by PCR, and subucloned into pET-28a(+) vector. Recombinant plasmid was expressed by E.coli BL21(DE3), and the recombinant protein was purified by liquid chromatography. Results A 358 bp cDNA fragment was amplified by PCR method. Its structure between the ATG and TGA was completely consistent with the human sorl1 cDNA fragment(5 923~6 260 bp). The recombinant plasmid in E.coli BL21(DE3) was highly expressed and its expression product was mainly in the inclusion body. The purified protein on SDS-PAGE demonstrated a single band, which appeared to have a molecular weight of about 13 000. Conclusion Human sorl1 cDNA fragment was highly expressed in prokaryotic cells. The purified recombinant protein can be used to prepare antibodies.