Cloning, Characterization And Expression Of TIP30 Genes From Amphioxus Branchiostoma Belcheri Tsingtauense

TIP30, also called CC3 , is an evolutionarily conserved gene that is expressed ubiquitously in human tissues, TIP30 plays an important role in suppressing metastasis of carcinomas and enhancing transcription of HIV-1 Tat. Amphioxus or lancelet, a basal chordate, is the closest living relative to vertebrate, and has been widely known as the most important animal to study the origin and evolution of vertebrates. Studies on the gene structure, function and expression in amphioxus will contribute to the understanding of the origin and evolution of the vertebrates.In this paper, the antimetastatic gene, AmphiTIP30 has been cloned by the methods of RT-PCR and RACE. The full-length cDNA of AmphiTIP30 is 1499bp and it contains an open reading frame of 822bp. There are two initiation codon (ATG) in the 5'-end: The first initiation codon was to encode signal peptide which consisting of 23 amino acids. The second initiation codon was to encode the mature peptide which comprises 237 amino acid residues with a predicted molecular mass of approximately 26 kDa. The deduced amphioxus protein has 21.3–28.7%, 17.3-41.3%, 53.2%, 49.8%, 50.6-51.9% identity with TIP30s of microbes, invertebrate , teleost ,frog and mammals, respectively, and contains the 3 conserved residues (Ser166, Tyr177 and Lys181) that are well conserved in the other TIP30 proteins. Phylogenetic tree analysis showed that vertebrate and invertebrate TIP30 proteins were each clubbed together, with AmphiTIP30 falling at the base of vertebrate TIP30 clade suggesting that amphioxus is a invertebrate most closely related to the proximate invertebrate ancestor of vertebrates.The recombinant TIP30 of amphioxus has been produced and purified. The molecular weight was estimated with 29KDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding constant and enzymatic activity was confirmed by biochemical assay. Western blot with TIP30 antibodies showed that the antibodies also reacted with the protein we obtained. In addition, the immunohistochemisty showed that AmphiTIP30 was mainly localized in the ovary, which indicated the TIP30 may have some relation with the origin of ovary.