Beijing Huaan Kechuang Biotechnology Co., Ltd.

The recombinant human nerve growth factor (rhNGF) with high yield was expressed by using Chinese hamster ovary (CHO DG44) cell line.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 polled 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 chromatog. and size-exclusion chromatog. 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 referenceAfter 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 com. available mouse NGF products.This study made solid foundation for the large-scale industrial production of rhNGF.

A nerve growth factor (NGF)-dependent TF-1 subclone cell line suitable was screened for determination of bioactivity of recombinant human NGF (rhNGF).The rhNGF-dependent TF-1 cells were habituated to mNGF-dependent ones by substitution of medium containing recombinant human granulocyte macrophage-colony stimulation factor (rhGM-CSF) with that containing mouse NGF (mNGF).The dose-response curve of mNGF-dependent TF-1 cells was plotted, based on which the ratio of signal to noise (S/N) and maximal effect (Emax) interval were calculatedThe mNGF-dependent TF-1 cells were subcloned with semi-solid Me cellulose medium, and the single clones were selected under microscope for amplification.The dose-response curve of various single clones against NGF was plotted, and the single clone with the highest reactivity was subjected to STR profiling.The d. of cells inoculated and the time for culture were optimized, and the screened cells were used for determination of bioactivity of rhNGF.After habituation, rhGM-CSF-dependent TF-1 cells grew normally in the medium containing only mNGF, and showed good dose-response to the stimulation with NGF.The S/N and Emax of dose-response curve of mNGF-dependent TF-1 cells against mNGF were 2.0 and 0.43 resp.Fifteen single clones with good morphol. and stable amplification time were screened, of which TF-1-A2 showed the highest reactivity.TF-1-A2 showed no cross contamination with other human-derived cells but only a difference in CSF1PO loci from that of TF-1 cells deposited in ATCC, which was judged as TF-1 cells.The optimal d. for inoculation and time for culture were 5×10⁴ cells/mL and 72 h resp.Under the optimal condition, the cells showed good reactivity and might be used for determination of bioactivity of rhNGF.TF-1-A2 cell strain with good reactivity to NGF was successfully screened, which was suitable for the quant. determination of bioactivity of NGF.