基因重组型人SORL1蛋白质片段的原核表达和纯化

Prokaryotic expression and purification of recombinant human SORL1 protein

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。

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.