The Construction And Identification Of Recombinant Human NGF Expressed In HEK293 Cells

Nerve growth factor(NGF) is the earliest discovered nerve cell growth regulating factor and extensively studied, which has dual biological functions of providing nutrition for the neuron and promoting neurite growth. In recent years, researches discover that NGF are not only confined to the aspect of central and peripheral nervous system s, but also play active roles in the treatment of bedsore, corneal ulcer, glaucoma and immune diseases. At present, mouse NGF is most widely used in clinic, however, its production method and product defects should not be ignored. For example, it is easy to cause allergic reaction and induce antibody response, may be contaminated with the virus; production process is complex and can not be automated, high production costs and medicine prices, which draws our attention. On the other hand, although the researchers try to develop recombinant human NGF beta subunit(rh-β-NGF) through a variety of methods and the production technology of recombinant drugs from different systems, as adoption of prokaryotic expression system and insect system different from the human species, they can’t obtain high biological activity of rh NGF; or even using the eukaryoticexpression system, but the lack of complete recombinant vector led to relatively low production efficiency. Therefore, we firstly tried to construct a stable and efficient eukaryotic expression vector of human NGF, then add the regulatory elements of transcription and translation, replace the signal peptide sequence, finally detect the expression level of NGF and identify its biological activity to lay the foundation for the large-scale production of NGF.1. Construction of recombinant human NGF expressed in HEK293 cellsFirstly, the recombinant plasmid pCMV-β-NGF-IRES-dhfr was constructed, it consists of five parts: the promoter(CMV), beta globin gene intron, human beta-NGF gene and internal ribosome entry site(IRES) and selective marker gene(DHFR). Then the recombinant plasmid was transfected into HEK293 cells and the positive cells were selected by MTX pressure screening and limited dilution method to obtain monoclonal recombinant cell line efficiently expressing rh NGF. The recombinant cells were the same as ordinary HEK293 cells in morphology, the rate of recombinant cells growth and apoptosis was similar to primary cells, no obvious differences were observed. Subsequently, we gradually decreased the concentration of serum in medium to completely adapt cells to serum-free medium and stably express rh-β-NGF(namely rh NGF. In this article, rh NGF are all referred to rh-β-NGF.) Analyses of the expression products by SDS-PAGE noted that there was a visible band with relative molecular mass of about 13 k Da and the purity of more than 50%, the peptide mapping was tested by mass spectrometry and was matched theoretical sequence, then we used SP Sepharose FF strong cation exchange chromatography to purify rh NGF with the purity of more than 95%; Finally, the expression efficiency and stability of recombinant cell line was detected, the results showed that after sixty passages, the recombinant cell strain still stably growed and efficiently expressed rh NGF with the secretion of more than 20 pg/cell?d.2. Identification of recombinant human NGF activityBiological activity of rh NGF has the value of clinical application, therefore,detection of rh NGF is an important part in this paper and is one of the basic works in our group.PC12 cell line is differentiated from a rat adrenal medullary chromaffin tumor, has the general characteristics of neuroendocrine cells, due to its passage culture, it is widely used in research on neurophysiology and neuropharmacology, the detection of NGF biological activity by induction is a classical experiment. In this experiment, as PC12 cells grew to the density of 90%, m NGF and rh NGF were added to cells by a final concentration of 5 μg/m L, PBS was used as negative control. The results revealed that compared with the negative control group, cells could grow more fibrous projections in rh NGF group, which suggested that rh NGF secreted by the recombinant cells could induce the differentiation of PC12 cells.Chick embryo dorsal root ganglia(SCG) tissue culture method is commonly used experimental to detect NGF biological activity. SCG of 8 d fertilization chick embryo was dissected and cultured in Petri dish, when the ganglion adherently grew, the medium was replaced with the fresh culture medium, and m NGF and rh NGF were added to cell suspension. Cell migration was observed in each group after 24 h. More cells emigrated and formed neural processes in rh NGF group after 48 h, and the similar phenomena were also observed in m NGF group. The emigrated cells grew well in rh NGF and m NGF groups, but they became out of order and apoptotic cells existed in the PBS group. These findings revealed that m NGF and rh NGF could provide nutrition for neuron and promote neurite growth.Then newborn Kunming mice were used and treated with rhNGF, and the expression of Trk A in cervical skin of Kunming mice was detected by Western blot. We found that the expression of Trk A were similar in rh NGF group and m NGF group, compared with the negative control group, and the expression of Trk A significantly increased in the two groups. Meawhile, there were no differences in the expression of Trk A on both sides of the neck, suggesting that rh NGF not only play a role at the injection site, the upregulation of Trk A also occurs in the skin away from the injection site. Theoretically, rh NGF should work through the body.The superior cervical ganglion of neonatal rats(SCG) was dissected, short diameter(a) and long diameter(b) of SCG were measured by the microscope, and the volume of SCG was calculated by the method of 1/2×a × a × b and analysed. The results showed that the volumes of SCG significantly increased in rh NGF group and m NGF group compared with the control group. The analysis of the bilateral volumes of SCG suggested that there was no difference in bilateral SCG volumes of neonatal mice in the same group. The treated mouse SCG was sectioned and stained by toluidine blue, which indicated that SCG neurons were hypertrophy in the two NGF groups compared with PBS group.Mast cells can secrete nerve growth factor which combines with specific NGF receptor in mast cell membrane, thus mast cells are activated and degranulated by the second messenger Ca2+, release allergic media and histamine which work in nerve endings to stimulate the release of neurotransmitter. The morphology of mast cells of treated neonatal rat skin was analyzed,and the mast cell clustering and degranulation were found in the two NGF groups, which showed that rh NGF could work by combining with specific receptor of mast cell.In short, the recombinant cells can efficiently and stably exprese rh NGF and its activity is similar to that of medicinal m NGF in vitro and in vivo. Therefore, we will further study the r-β-h NGF application in clinic.