重组人神经生长因子的高效表达和生物活性评价研究

Study on the High Level Expression and Bioactivity Evaluation of Recombinant Human Nerve Growth Factor

目的利用中国仓鼠卵巢(Chinese Hamster Ovary,CHO)细胞真核表达系统,获得高产的人神经生长因子(recombinant human Nerve Growth Factor,rhNGF)表达细胞株并对其分泌的rhNGF生物学活性进行评价。方法设计、合成并构建了携带rhNGF目的基因的GS-DHFR双基因筛选表达载体pDG2.0/NGF。经脂质体转染法将其导入二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞(CHO DG44)中,经氨甲喋呤(MTX)和蛋氨酸亚氨基代砜(MSX)两种药物加压筛选和克隆化获得目的细胞株。经过阳离子交换和分子筛纯化,获得rhNGF制品。最后,利用小鼠神经生长因子生物学活性参比品评估纯化制品的生物学活性。结果通过基因合成、载体构建、细胞转染、加压筛选和克隆化等步骤,我们得到了高表达rhNGF的CHO-DG44单克隆细胞株。经过2步法纯化获得了纯度大于98%的精制rhNGF制品。生物学活性分析表明,与市售小鼠NGF产品相比较,我们所表达的rhNGF具有更好的生物学活性。结论成功实现了rhNGF的高水平和高活性表达,为大规模工业化生产重组人神经生长因子奠定了坚实基础。

Objective To express recombinant human nerve growth factor （rhNGF） with high yield using Chinese hamster ovary （CHO DG44） cell line. Methods In brief, rhNGF gene was optimized, synthesized and subcloned into the pDG2.0 expression vector co-expressing GS and DHFR proteins. Then, the resultant plasmid pDG2.0/NGF was transfected into CHO-DG44 cell by lipofectin. After transfection, the pooled transgenic CHO DG44 cells were cultured and selected. Separated single clone with high yield of rhNGF was obtained by semi-solid medium culture, and rhNGF was purified by ion exchange chromatography and size-exclusion chromatography from the culture supernatant of the said single clone. The bioactivity of purified rhNGF was evaluated by Dorsal root ganglia （DRG） method and TF-1 cell proliferation method with mouse NGF standards as reference. Results After transfection of CHO DG44 cells with plasmid containing optimized human NGF gene, the stable monoclonal cell lines expressing rhNGF with high yield were obtained by subcloning with semi-solid medium method. The expression level of rhNGF expressed in said transgenic CHO DG44 cells was tested and verified by Sandwich ELISA and Western blot. After two-step purification, rhNGF with purity 〉 98% was harvested, then its bioactivity was evaluated with mouse NGF reference material. The bioactivity of purified rhNGF was much better than commercially available mouse NGF products. Conclusion This study makes a solid foundation for large-scale industrial production of rhNGF.