【摘要】 目的 :制备有活性的 Trk A膜外域各结构域重组蛋白。 方法 :用 PCR法克隆 Trk A膜外域各结构域 (分别命名为CL C、Ig1和 Ig2 )的 c DNA片段 ,然后插入融合表达载体 p ET- 2 8a(+)中 ,经测序证实后 ,转入大肠杆菌 BL2 1中表达 ,并用亲和层析法进行纯化 ,用神经生长因子 (NGF)诱导 PC12细胞株测定 Trk A与配体结合的关键结构域 (CL C、Ig1和 Ig2 )活性。结果 :成功地用大肠杆菌制备了纯度达 90 %以上的 Trk A膜外域各结构域重组蛋白 ;NGF+Ig2组 PC12细胞突起生长率明显低于 NGF组 (P<0 .0 5 ) ,NGF+CL C、NGF+Ig1组与 NGF组相比没有显著差异。 结论 :Ig2能抑制经 NGF诱导的 PC12细胞的分化 ,CL C和 Ig1不能抑制 PC12细胞的分化更多还原

【Abstract】 Objective: To obtain biologically active recombinant TrkA extracellular domain proteins.Methods: The cDNA encoding TrkA extracellular domains (CLC,Ig1 and Ig2) were amplified by PCR and the products were inserted into the expression vector pET 28a(+).The expressed proteins were purified by affinity chromatography and were tested with PC12 cells.Results: The high purity(90%) recombinant fusion proteins were prepared by E.coli .The activities of key TrkA binding domain(CLC,Ig1 and Ig2) to ligand were confirmed by PC12 cells.There was significant difference between NGF group and NGF+Ig2 group in their ability to induce neurite outgrowth( P <0.05 );there was no significant difference when comparing NGF+CLC and NGF+Ig1 group with NGF group.Conclusion: Ig2,but not CLC or Ig1,is able to inhibit neurite outgrowth of PC12 cells.更多还原