重组人神经生长因子rh-β-NGF真核表达载体的构建及其在HEK293细胞中的表达

Construction of recombinant human nerve growth factor (rh-β-NGF) eukaryotic vector and its expression in HEK293 cells

摘要:

为大量制备β-NGF，构建了一种稳定、高效表达重组人神经生长因子 (Recombinant human nerve growth factor, rh-β-NGF) 的真核表达载体及含该重组载体的HEK293细胞株。首先，构建重组质粒pCMV-β-NGF-IRES-dhfr并转染至HEK293细胞系，用MTX加压筛选和有限稀释法进行选择，获得高效表达rh-β-NGF的单克隆重组细胞株；随后逐步降低血清培养，最终使细胞株完全适应无血清培养基并稳定表达rh-β-NGF；SDS-PAGE分析该表达产物，可见相对分子质量约13 kDa的条带，纯度大于50％，经质谱法测定得到其肽图谱与理论序列完全匹配，接着利用离子交换层析和分子筛层析纯化rh-β-NGF；最后进行重组细胞株表达效率和表达稳定性检测，表明重组细胞株可稳定、高效表达rh-β-NGF，其分泌效率大于20 pg/(cell?d)，并能诱导PC12细胞的分化，具有良好的生物学活性。

Abstract:

Human nerve growth factor (NGF) is a nerve cell growth regulation factor, which can provide nutrition for the neurons and promote the neurites outgrowth. In order to produce large-scale recombinant human nerve growth factor (rh-beta-NGF), we constructed a plasmid vector, which can stably express the rh-beta-NGF in the HEK293 cell lines. First, the plasmid of pCMV-beta-NGF-IRES-dhfr was constructed and transformed into HEK293 cells. Then MTX pressurized filter and limiting dilution methods were used to obtain monoclonal HEK293 cell lines. After stepwise reducing serum in culture media, the cells eventually adapted to serum-free medium and secreted rh-beta-NGF. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 13 kDa and a purity of more than 50%. The peptide mapping sequencing analysis demonstrated the sequences of rh-beta-NGF matched with the theoretical ones. Later we purified this protein by ion exchange and molecular sieve chromatograph. Finally, our experimental results exhibited that the recombinant cell lines can stably express rh-beta-NGF with a high efficiency of more than 20 pg/cell?day. In addition, this protein could successfully induce differentiation of PC12 cells. In summary, our recombinant HEK293 cells can express bio-active rh-beta-NGF with great efficiency and stability, which supply a valid basis to large-scale production of rh-beta-NGF.