Studies On Preparation, Bio-assay And Transmembrane Of PTD-NGF Fusion Protein

Nerve growth factor was discovered as the first neurotrophic factor. The natural NGF is composed of 3 subunits (α,βandγ) and only theβsubunit has biological activity.β-NGF is a homodimer and each subunit contains 118 amino acids. NGF is necessary for nerve cell growth and cytothesis. It also has ability on regulating interaction between immune system and endocrine system.Even more, it can promote wound healing, cell growth and inhibit tumor cell amplification. NGF possesses evident therapeutic effects upon Alzheimer's disease which is caused by cholinergic neuron decline, newborn infant ischemia cerebropathia and Parkinson's disease. Therefore, NGF plays important role in basic and clinical research on nerve repair and regeneration.Now, NGF drug is applied to clinical patient for peripheral neuropathy. Because of blood brain barrier, the applications of NGF to central nervous system disease are limited.Trans-activating protein (TAT) was expressed by AIDS virus. In the molecule structure of this protein, there is a length of polypeptide with rich alkalinity amino acids (TAT48-60). The polypeptide which is called protein transduction domain (PTD) can mediate protein transport into cell. PTD is able to deliver many kinds of bio-macromolecule across bio-membrane, even can across blood-brain barrier with no harm and there is no apparent size restriction to this process. So, there are extensive applications of PTD in the preclinical and clinical medicine.On account of above reasons of PTD, PTD-NGF fusion gene was constructed and biological function and transmembrane ability of the recombination protein was verified. The main content and result of my study is showed as follow:1. Construction of prokaryotic expression vector of the PTD-NGF fusion geneAfter extracting RNA from mouse brain tissue, the cDNA Sequence coding forβ-NGF was cloned from mouse brain RNA by reverse transcription. NGF gene was amplified by PCR and inserted in the cloning plasmid pcDNA/FRT. The PTD gene sequence was fused to the 5′end of primer for NGF, and then, the PTD-NGF fusion gene was amplified by PCR from the plasmid pcDNA/FRT-NGF. At last the PTD-NGF fusion gene was inserted in the expression plasmid pBV220, and identified by enzyme digestion assay and sequencing.2. Expression of the PTD-NGF fusion geneThe expression plasmid pBV220/PTD-NGF was introduced into the competent cells of E.coli.DH5α, which were induced at 42℃to express the PTD-NGF fusion proteins.The expressed product was verified by SDS-PAGE and Coomassie brilliant blue dyeing. The molecular weight of expressed product was about 14×103, same as PTD-NGF fusion protein. The fusion protein mainly existed in inclusion bodies, reached about 20% of total cell proteins, and 70%of total inclusion bodies.3. Purification, renaturation and identification of the fusion proteinThe inclusion bodies after fully washing and dissolving by urea, was purified through Sephacryl-200 gel chromatography column. The main protein peak was verified as the PTD-NGF fusion protein by SDS-PAGE. The dissolved protein was re-natured by dialyze urea step by step. The renaturation protein was condensed through PEG, and its concentration was 300ng/ml by Bradford assay.The recombantant protein was authenticated by Western-blotting assay.4. Exploration of bioactivity of PTD-NGF fusion proteinPC12 cells were used to analyze its biological function after cultured with PTD-NGF fusion protein. Two weeks later the cells became larger and more irregular, and had more enations comparing with the control group. It was concluded that the fusion protein could promote the differentiation of PC12 cells as NGF.HepG2 cells were used to detect its transmembrane ability by immunohistochemistry. After incubated with PTD-NGF fusion protein, the fusion protein could be detected in the HepG2 cells with anti-NGF antibody, while comparing with the control group.It showed that the fusion protein had the ability of transmembrane transport.In short, the PTD-NGF fusion gene was constructed and expressed in E.coli successfully.The fusion protein was purified and renatured, which had the biological function as NGF, and the ablity of transmembrane. The research makes a sound foundation in application of NGF on therapy for nerve injury and degeneration diseases of nervous system.