| Scorpion anti-neuroexcitation peptideⅢ(ANEPⅢ) of Buthus martensii Karsch (BmK) has better antipileptic and anticonvulsive effects in the test animal models in vivo and in vitro. The results of conventional pharmacological and acute toxicity tests also indicated that this polypeptide was quite possible to be the first clinical peptide medicine of the prophylactico-therapeutic pharmaceutics for brain neuroexcitopathy.In the present study, the transgenic plant expression system was applied to obtain complete modified and dissoluble protein, BmK ANEPⅢ. ANEPⅢgene was aimed to obtain over-expression in plants. Using molecular cloning technique, ANEPⅢgene was inserted (added Kozak sequence to 5’end and the ER retention signal to 3’end) into vector pBin438 to construct plant expression vector pBin-ANEPⅢ. The ANEPⅢexpression cassette included CaMV DE35S promoter,Ω’enhancer, ANEPⅢand NOS terminator. Based on pBin-ANEPⅢ, the pBI121-ANEPⅢwas also construed successfully. Recombinant plasmids were transfered into A.tumefciens EHA105 by electroporation and freezing transformation methods, and pBin-ANEPⅢ/EHA105 and pBI121-ANEPⅢ/EHA105 were obtained by verifying.Tobacco (Nicotiana tabacum) and tomato(Lycopersicom esculentum) were transformed by Agrobacterium-Mediated leaf disc transformation method. Transformants were screened to grow and root on media containing kanamycin. Then, transformations were confirmed by analysis of PCR, RT-PCR and western blotting, and the results confirmed that the ANEPⅢgene in transgenic plants genomic DNA was successfully expressed. The expression level of the ANEPⅢprotein was respectively estimated up to 0.81% and 1.08% of total soluble proteins in transgenic tobacco and tomato by analysis of immunofluorescence technique. The study has important theory significance and application value through plants expressing ANEPⅢ, and it will provide practice information for establishing new, cheap and large ANEPⅢproduce route. |
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