| Background and objective:Human society is facing to a difficult problem, an aging population, all over the world. Accoding to the newest statistics data of World Health Organization, the proportionality of the crowd exceed 60 years old will be from 11% in 2006 increasing to 22% until 2050. In our country, as early as 2003, the crowd exceeded 60 years old had surpassed one hundred and thirty-two million, and the proportionality is 10% of the whole population, we has been stepped into an aged period, a big aged crowd will have heavy effect on our society, economy and culture. In order to make preparation for the persistent development of society with a rapid rise of an aged population, the prevention and cure of gerontal disease has no time to delay. Alzheimer's disease ,characterized clinically by progressive dementia, is the most frequent nerve degradation disease which happened on gerontism or ahead of it. Its principle clinical symptom is progressive loss of memory ability and cognitive impairment, personality change and cortex abruption, loss of self-possession and perceptivity, and in advanced stage, memory, linguistic and behavior capacities of patient will completely lossed. The course of disease is about 10 years, the patient will become plant man finally. The characteristics of AD on pathology are to form senileplaques(SP), neurofibril tangles(NFT), and neuron loss. Because the primary pathogenesis and cause of AD is not yet clear and lack of effective early diagnostic and therapeutic methods, AD has already been the fourth leading cause of death after heart disease,cancer and stroke. Therefore researching AD, postponing the appearance of AD, and ameliorating the life quality of the patients is a pressing research project. Up to now, we know a little about the pathogeny and the pathogenesis of AD, so the therapy for AD is intractable. Although the deprivation of cellula nervosas is nonreversible, drug treatment could conduce to delay the development of the disease all the same. Acidic peptide(AP) which is a kind of small biologically active peptide is isolated from bovine brain and distributs widely in the central neural system of animal. Experiments in animal model have proved that:①Acidic peptide has the effect of antioxidation;②By increasing the synthesis of protein, AP can promote the recovery of brain damage.③AP can promote the expression of Bcl-2, inhibit the expression of Bax and the generations of NO,βamyloid peptide and NMDA receptor, for this reason AP can restrain the apoptosis of neuron.④AP can promote the synthesis of and release NGF and BDNF, and promote asteroid cell multiplication, and increase the survival rate, AP has the effect of nutrition and protection to the growth of asteroid cell and neuron.⑤Acidic peptides is a small molecular neuropeptide without antigenicity, and can permeate blood brain barrier. However, acidic peptide from animal's brain has the chance of bring the factor of mad-cow disease and various virus. It cannot be used directly in clinic. Therefore, this topic synthesized assembled gene clone of acidic peptide and succeeded in expression research work foundation in Escherichia coli., the mass culture this host fungus, the induction assembled acidic peptide gene's protein expression, and carries on the separation and the purification to it, with the aim of producing the safe reliable non-animal source the acidic peptide, provides the safe effective drug for the clinical care of senile dementia.Methods:Used IPTG which final concentration was 1.0mmol/L to induce the host bacterium M15(pREP4) which contained the pQE30-assembled gene of acidic peptide to express the fusion protein, centrifuged and collected the bacterium, spallated the bacterium with two methods, supersonic spallation and the water boils spallation, then make the bacterium lysate into powder through freeze concentration, dissolves the powder into 10% solutions, filtered it, collected the clear supernatant liquid, used Sephadex G-25, G-50, G-75 three kind of gelatin to carry on the purification and separation separately, collected eluent, and with the SDS-PAGE gel electrophoresis analyzed the target protein's purification and separation situation, according to the electrophoresis results, made the amino acid appraisement to the related eluent samples.Results:1. 10% bacterium spallation supernate after Sephadex G-50 on column purification, separates His-assembled acidic peptide fusion protein the effect to be best.2. After the Sephadex G-50 purification, the fraction collection eluent were made SDS-PAGE gel electrophoresis analysis, the 40-50th tubes contains the protein ingredient to be the most unitary.3. Makes the amino acid appraisal to the 40-50th these 10 eluent, proved that contains the amino acid for the the glutanic acid, is acidic peptide fusion protein.Conclusions:In many kinds of protein which this experiment (pREP4) after passing through IPTG induction expresses from pQE30- combination acidic peptide gene clone's host fungus M15, purified using Sephadex G-50 separates the molecular weight approximately is the 4.5KD His-combination acidic peptide fusion protein successfully, and after amino acid appraisal confirmation. |
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