Cloning And Prokaryotic Expression Of Growth Associated Protein-43 In Bufo Gargarizans

The ability of neural development and neural regeneration of vertebrate central nervous system is different among fish, amphibians and mammalian. The reason causing regenerative ability different is that molecule in microenvironment of neural regeneration is discriminating in different animalia. As a representative molecule in neural development and neural regeneration, the primary structure of GAP-43 has variated in evolution of vertebrate. It is significant that research the difference of Bufo gargarizans's GAP-43 with other vertebrate to vertebrate central nervous regeneration.In this research, take Bufo gargarizans's GAP-43 as object of study, designed primers according to the cloning principle of homologous sequence. Cloned successfully Bufo gargarizans GAP-43 gene by RT-PCR, the lengh be sured is 654bp by sequencing. The accession number in Genbank is FJ794607. Translating the coding sequence into acids by software, and making acids sequences analyzing of GAP-43 among fish, amphibians and mammalian, in total 13 species. The result illustrates that the 3,4 Cys site, "IQ" Motif, phosphorylation site of PKC in GAP-43 acids sequence is conservative, but the sequence homology is lower from 66 site to C end, and the GAP-43 sequence of Bufo gargarizans GAP-43 has more SerMaking predictions of hydrophily, signal peptide, transmembrane domain, subcellular localization and secondary structure to Bufo gargarizans GAP-43 acids sequence. The result illustrates that Bufo gargarizans GAP-43 acids sequence displays acidity and hydrophily. signal peptide is not existence in it's N end, transmembrane domain also is not existence and is not a transmembrane protein.subcellular localizate to cytoplasmic membrane of nerve cell.Bufo gargarizans GAP-43 acids sequence has 18 secondary structures, Coils is 50%, Helixs is 35% and Strands is 25%.In this research, we structure successfully the Recombinant plasmid of pET32a-GAP-43,then transform E.coliBL21 and abducte expression, Bufo gargarizans GAP-43 protein with tag can be expressed by SDS-PAGE electrophoresis detection. And gained high purity quotient fusion protein by Ni-Resin HP method.