Expression Of Human Nerve Growth Factor In Cho Cells With Various Vectors

Nerve growth factor (NGF) is the first discovered neurotrophic factor. It has an effect on nervous system as well as non-neuronal systems. And NGF is widely used to treat a variety of nerve diseases. Three kinds of eukaryotic expression vectors were constructed , a number of transcription and translation regulatory elements were added, the signal sequence was replaced, and the expression levels of human NGF in CHO cells with the three recombinant plasmids were compared, the recombinant plasmid most highly expressing human NGF was screened, which lay the foundation for large-scale production of NGF.The three recombinant plasmids were pMD902-β-NGF, pcDNA3.1-His-β-NGF and pcDNA3.1-4.0-His-β-NGF. Compare them with pcDNA3.1-β-NGF in NGF expression and biological activity. pMD902-β-NGF: The coding region ofβ-NGF gene was digested by Sal I from pcDNA3.1 -β-NGF, and the gene fragment was inserted into plasmid pMD902.pcDNA3.1-His-β-NGF: The coding region ofβ-NGF gene was amplified by PCR from the template pcDNA3.1-β-NGF. Transcription regulation region-8×His-EK (enterokinase) site was inserted into the new plasmid in the process of PCR amplification by designing three pairs of primers. In the process the signal peptide of NGF was instead of the signal peptide of IgG kappa chain.pcDNA3.1-4.0-His-β-NGF: The translation regulatory sequence from plasmid pcDNA4.0 was synthesised chemically and inserted into plasmid pcDNA3.1 between HindⅢand BamH I to construct a new vector pcDNA3.1-4.0, and then, the gene fragment of transcription regulation region-optimized signal peptide-8×His-EK (enterokinase) site-NGF was inserted into this vector, the resulted vector was named pcDNA3.1-4.0-His-β-NGF.The recombinant plasmids pMD902-β-NGF , pcDNA3.1-His-β-NGF, pcDNA3.1-4.0-His-β-NGF and pcDNA3.1-β-NGF were transfected into Chinese Hamster Ovary cell (CHO cell) respectively. Expression of NGF in CHO cultural supernatants was confirmed by ELISA after screening. It shows that the four all can express NGF protein. The expression concentration of pMD902-β-NGF is about 0.6ng/ml , pcDNA3.1-β-NGF is 4.7ng/ml, pcDNA3.1-His-β-NGF is 17.7ng/ml, and pcDNA3.1-4.0-His-β-NGF is 28.3 ng/ml.Biological activity of the expressed human NGF was measured by the test of its simulating nerve fiber growth of chicken embryo DRG neurons. They can all stimulate the dorsal root ganglia grow around the nerve fibers. This showed that the expressed human NGF has good biological activity.