The Preparation Of The Polyclonal Antibody Of The Anti-epilepsy And The Detection Of RAAV/AEP

Background: The scorpion has been used to treat epilepsy disease as a kind of drug by practitioner of Chinese medicine for thousands of years. The view is accepted by most scholars that the main active constituent is scorpion toxin. Anti-epilepsy peptide(AEP),one of the scorpion toxins, can treat epilepsy. The fact has been verified by some experiments. Also, the complete amino acid sequence of it has been determined. And the anti-epilepsy effect of AEP was proved in experimental models of epilepsy.Various tissues have been successfully transfected by rAAV vectors with no detected toxicity. And successful rAAV-mediated gene deliveries have been reported by many scholars. Now, gene therapy has become one hot spot. Professor Wang zong-ren have got the gene of AEP successfully from Asian scorpion Buthus martensi Karsch,and cloned it to AAV2 vectors. The fact that rAAV/AEP had been founded can provide a new way in gene therapy of epilepsy. AEP is a peptide, whose molecular weight is 8.3 KDa. The way to detect it simply and reliably is absent now. And there is no report about the successful preparation of the antibody of AEP. If we could get the antibody of AEP, the more specific method to detect AEP would be founded, and it would become the base to further study of rAAV/AEP.Methods: The vector pGEM-T/AEP was digested with BamH I/EcoR I ,then, the product was cloned into the expression vector PGEX-4T-1 after the restriction sites was changed finally. The protein GST-AEP was expressed in E.Coli-DH5αas a fusion protein induced by IPTG. The fusion protein was identified by 12% SDS-PAGE. The purifying and refolding to inclusion body were optimized. The purity of GST-AEP was identified by 12% SDS-PAGE and thin-layer scanning analysis. The quantitation of the fusion protein GST-AEP was done with BCA Protein Assay. The fusion protein was given to rabbits by hypodermic injection. The antibody in blood serum of rabbits was determined by ELISA and western-bolt. The rAAV/AEP was transduced to 293 cell line and rats. The gene transduction was identified by RT-PCR assay.Results: AEP coding region was cloned into pGEX-4T-1 vector and the sequence was confirmed to be right. The fusion protein GST-AEP was correctly expressed and identified as inclusion body. The purity of GST-AEP was higher than 90% and concentration was about 0.163μg/μl. The antibody of GST-AEP was obtained , the titer of antibody was determined as 1:12000 and the sensitivity of Antibody was about 0.065μg. The polypeptide AEP was only detected in the medium without serum but not found in serum of rats. The gene AEP could be detected in muscle of experimental rats.Conclusion: The fusion protein GST-AEP was obtained successfully by the prokaryotic expression. The polyclonal antibody of AEP was prepared and identified. The activity of rAAV/AEP could be found. The reason why AEP could not be detected in serum of rats may be the concentration of secreted AEP is too low.