Screening The Target Molecule Interaction With AEP And Exploring The Anti-epileptic Function Of AEP

Epilepsy is one of the most frequently nervous system diseases. It was reported that the prevalence of epilepsy was 7‰, and annum incidence was 28.8 per 10 thousands people in China. There are almost 9 million epilepsy patients in China,which accounted 1/5～1/6 of total patients all over the world.The number of new cases is 4 million a year. Epilepsy is a disease that has high mutilation, iteration attack on clinical and a lengthy course. Once the diagnose of epilepsy was validated, the patient should have the drug in time, and are demanded to take the medicine for a long-term (majority patients have taken it more than 3 years), so it is very easy to cause drug resistance, meanwhile the anti-epileptic drug has many side effects, such as stimulation of gastrointestinal tract, dysfunction of liver, hypofunction of remembrance and recognition, and so on. All of this has constrainted the clinical application and therapeutic effect of itself. About 25%～30% of patients have become to refractoriness epilepsy. It threatens the physical and mental health of patients, affects the life quality of patients and aggravates the economic burden. To seek a more safe and utility treatment method of epileptic is a hot spot of astrology research. Anti-epilepsy peptide is a polypeptide which has biologic activities against epilepsy. It was abstracted from the venom of Buthus martensii Karsch(BmK),and has favorable effect of anti-epileptic. Its therapeutics mechanism is still unclear at present. To investigate the therapeutic mechanism of the epileptic of AEP, gene recombination technique was employed to construct the expression vector of procaryon and eucaryon, derivate recombination protein expression, and rat model of epilepsy was duplicated. Molecule interaction with AEP was detected by pull-down experiment, and analyzition of the target protein by bioinfomatics to reveal the therapeutics mechanism of AEP. This would be helpful to develop new anti-epileptic drugs and also can provide new ways for the research of traditional Chinese medicine mechanism of action.Our finding can be sum up to four parts below:1.We have constructed the sequencing vector PUC18-T-AEP and prokaryotic expression vector pGEX-4T-1-AEP ,all of which have the AEP gene fragment after the DNA sequencing; take the recombination expression vector pGEX-4T-1-AEP to in the cells of BL21(DE3) competence Escherichia coli, we procure the high performance express after IPTG induced, then by the Western blot verification it is fusion protion;to hypersound schizolysis the express bacterium we find that the fusion protein is exist as inclusion bodies form;then depurate the inclusion bodies protein by glutathione sepharose 4B after ablution,urea denaturation and dialys renaturation.2. We have duplicated the rat model of KA epilepsy to prepare the homogenate of brain.To bolt the interact protein with AEP by GST pull-down test, after many times we have not achieved the expect purpose, it is considered that the AEP molecular mass is small but the molecular chaperones GST which confluent with the AET is big, so it has masked its effect binding site.3.We have constructed pPIC9K-AEP2-His the expression supporter of AEP by gene recombination technique,electrotransported it to SMD1168 , bolted out the hypso-copy converters by G418, carried out the PCR reaction by pPIC9K supporter, after identitied the AEP2-His gene integration from chromosome genome, it can express in the SMD1168 by methanol induced,then identified by the Western blot of anti-His monoclonal antibody; purified the AEP2-His proteinum by Ni-chelating Sepharose FF,then test the quantitation by BCA proteinum quantitation kit,take the SDS-PAGE verification.4.Reproduce the rat model of KA epilepsy, take out the hippocamp organize, then extract the membrane protein of hippocamp organize by Eukaryotic membrane protein extraction reagent kit, incubate it with the Ni-chelating Sepharose FF which combined with the AEP2-His protein, bolting the interaction molecule of AEP by His pull-down,at the same time set up compare model,the consequence manifested that: compared with the compare model that the AEP2-His protein incubation model has four strip conspicuous protein strip,take the four strip conspicuous protein strip out to take mass chromatographic analysis,the result indicate that the four kinds of protein are presynaptic membrane protein SNAP-25 (synaptosome-associated protein), GFAP(glial fibril-lary acidic protein), NMDAR (N-methyld-aspartate receptor) and GAD(glutamic acid decarboxyl- ase).So we presume that the four kinds of protein maybe is the target protein of AEP to produce anti-epileptic effect,then they produce the anti-epileptic effect by which molecule passageway to take the signal transduction between them is still need to research further.