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Expression of Human Nerve Growth Factor in Cho Cells with Various Vectors

Author: QiZuo
Tutor: LiYueXi
School: Nanjing Medical University
Course: Biochemistry and Molecular Biology
Keywords: Human nerve growth factor Clone Eukaryotic expression Biological activity
CLC: Q42
Type: Master's thesis
Year: 2009
Downloads: 27
Quote: 0
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Abstract


Nerve growth factor (NGF) is first discovered neurotrophic factor affects both the nervous system and nervous system, is widely used to treat the disease of nerve injury. In this study, by building the human nerve growth factor in several different eukaryotic expression vector, and increase the transcription and translation regulatory elements, the replacement of the signal peptide sequence, the expression levels of the human nerve growth factor, choose suitable for high expression of human NGF eukaryotic carrier, to lay the foundation for large-scale production of NGF. Build three recombinant plasmids are pMD902-β-NGF, pcDNA3.1-His-β-NGF and pcDNA3.1-4.0,-His-β-NGF was. Recombinant plasmid pMD902-β-NGF: pcDNA3.1-β-NGF, β-NGF gene inserted into the Sal I restriction sites of pMD902 vector. Recombinant plasmid pcDNA3.1-His-β-NGF: pcDNA3.1-β-NGF as a template to design primers for PCR amplification, before the β-NGF gene inserted into the period of transcriptional regulatory region sequences, 8 × His tag and EK (enterokinase) sites, the NGF signal peptide gene replacing the sequence of the signal peptide of the human IgG kappa chain gene sequences. That is ultimately the nerve growth factor gene and regulatory sequence (transcriptional regulatory region - to optimize the signal peptide -8 × His-EK (enterokinase)-NGF was) insert the vector pcDNA3.1 the BamH I and Xho Ⅰ sites. Recombinant plasmid pcDNA3.1-4.0-His-β-NGF: the section on chemical synthesis vector pcDNA4.0 translation regulatory sequences, inserted into the vector pcDNA3.1 the Hind Ⅲ and BamH I sites, build a new vector pcDNA3.1-4.0 The recombinant plasmid pcDNA3.1-His-β-NGF was nerve growth factor gene and regulatory sequence (transcriptional regulatory region - to optimize the signal peptide -8 × His-EK (enterokinase)-NGF was) to insert a new vector pcDNA3. 1-4.0 the BamH I and Xho Ⅰ sites. The three above-mentioned recombinant plasmid and plasmid pcDNA3.1-β-NGF transfected Chinese hamster ovary cells (CHO cells), the filter expression. A month later, ELISA detects the supernatant of cells transfected with the recombinant plasmid. The test results show that: the four recombinant plasmids can NGF expression. the pMD902-β-NGF expression about 0.6ng/ml pcDNA3.1-β-NGF was approximately 4.7ng/ml, pcDNA3.1-His-β-NGF was about 17.7ng/ml pcDNA3.1-4.0 -His-β-NGF approximately 28.3 ng / ml. Dorsal root ganglion assay detected four recombinant plasmid expressing the the NGF protein's biological activity, they can stimulate the dorsal root ganglion around the radial nerve fibers grow, indicating that the expression of nerve growth factor better biological activity.

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