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Therapeutic Effects of Human Epidermal Growth Factor Mutant Against Experimental Alzheimer’s Disease and Others

Author: ZhaoBaoQuan
Tutor: SunManZuo
School: PLA Military Academy of Medical Sciences
Course: Pharmacology
Keywords: hEGF mutant secretary expression Alzheimer’ s disease stomach injury skin health protection
CLC: R96
Type: PhD thesis
Year: 2007
Downloads: 74
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Abstract


Human epidermal growth factor (hEGF) is a single chain peptide consisting of 53 amino acid residues and three intrachainal disulfide bonds without free mercapto-radical. It has a molecular weight of 6045, and an isoelectric point of 4.6. In the C-terminus of the hEGF molecule locates the functional proliferation-promoting domain and on the N-terminus resides the binding domain. The hEGF can be biosynthesized in many tissues and organs, such as submaxillary gland, duodenum, kidney, pancreas, mammary gland, cornea, ovary, testis, prostate, liver, neuron and astrocyte, etc., playing important roles in protection of the cell functions. Clinically, the EGF are used for promotion of the healing of traumas, burns and scalds, for the repair of cell injury in the digestive and urinary tracts, and for the skin health protection and beauty treatment. However, the resources of the hEGF are limited. In the past, hEGF was extracted from the human urine with only a yield of 1g hEGF/105 L urine. There were also problems of inactivation and contamination. Recently, the biotech was introduced to production of hEGF, however hEGF often exsited in the form of inclusion body with no bioactivity and low yields. The denaturation and renaturation processes were complex and in low recovery. The cloning and expressing in large-scale of hEGF with stable biological activity was also very difficult. In our study, a secretary expression vector was successfully constructed with distinguishing features as follows: (1) There are only 5 nts between the OmpA initiation site and the binding site of ribosome nucleotides, thus it is favourable to the transcription and translation; (2) The series connection of OmpA and T7 promotor renders the target gene to large-scale and stable express in E.coli; (3) The uncharged glycine and serine residues added between the target peptide and OmpA make the molecule more flexible, favourable to the cleavage of OmpA by signal peptidase; (4) The OmpA signal peptide renders the exogenous gene product directly secreting into the periplasm, thus alleviates the cytotoxicity of the products expressed in the cytosol. (5) The protease activities in the periplasm are lower than that in the cytosol, so that is favourable to the stability of the fusion protein; (6) There are less contaminated proteins in the periplasm. The proportion of the expressed protein in total is higher than 60% as shown by the software analysis.In order to entrust novel features to the hEGF molecule, we fused the sequence of protein transdution peptide (PTD) to the N-terminus of the expressed hEGF mutant, so that it can permeate across cell membranes and even the blood-brain barrier. Thus, it opens the possibility of therapy against the neuro-degenerative diseases. It is known that hEGF can be expressed in neurons and astrocytes in the brain. EGF takes part in the proliferation, survival and migration of the precursor cells, and directly or indirectly plays a role in the regeneration of the neurons. EGF also exhibits neuroprotective actions to the cholinergic and dopaminergic neurons. EGF can secret NGF to promote the regeneration of neural cells via an indirect route of sustaining and inducing the growth of neurons and glia cells. EGF is also a kind of cerebro-intestinal peptide neurotransmitter. Ordinarily, EGF is difficult to penetrate through the BBB, a cerebral diffusion pump method is used in the experiments of brain research under the state of anesthesia. Unfortunately, it often inhibits the functions of the brain, and injures the brains of the animals from time to time. So it is difficult to mimic the real effects that acts upon the brains under the natural state of animals, and is not suitable for the clinical usage. The hEGF mutant expressed in our study is able to pass through the BBB, and lays the foundation of the therapy of diseases of the nervous system.The water maze and step through tests showed that the hEGF mutant could ameliorate the deficits in the Aβ-model mice and the aged mice. The PTD-hEGF could promote the proliferation of the stem cells in hippocampus. The micro-PET imaging showed that PTD-hEGF could stimulate the glucose metabolism in brain.Converging the data above-mentioned, it indicates that PTD-hEGF is effective in therapy of the experimental Alzheimer’s disease in mice and rats. Besides, it could also protect or remedy the alcohol-induced injury in the stomachs. The permeation capacity of PTD-hEGF across the skin and cell membrane is much better than that of hEGF per se. Thus the structure reconstruction of the hEGF molecule renders the possibility of the old drug for new usage in prospects of its practicality.The new approaches of this project are as follows:1. Successfully constructed the PTD-hEGF secretary expression vector, and well accomplished the soluble expression in prokaryotes.2. Successfully obtained the recombinant PTD-hEGF, a mutant with stronger permeation capacity across cell membrane than hEGF per se. Thus resolves the problem of permeation of hEGF across BBB.3. Opened up the route of administration of PTD-hEGF via rectum. Thus set up the base of sublingual preparation research and development for elevation of the safety of drugs and the obediencity of patients.

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