Primary Study On Expression, Functions And Biological Characteristics Of Pax6 Variant Protein Of Bufo Raddei Strauch

Pax6 is a very important transcription factor in eye development. It contains two DNA-binding domains (paired box and homeobox) and a praline-serine-threonine (PST)-rich transactivation domain. Pax6 gene is essential for the structural development of the eye and ocular cell differentiation in vertebrates.Pax6 gene has several mutations that have been found in the Pax6 in patients with Aniridia, Peter's anomaly, autosomal dominant keratitis, and familial foveal dysplasia.Twelve mutations have been detected in the PST region of Pax6, ten of them are truncation mutations.We made use of the tadpole of Bufo raddei Strauch at gills blood circulation stage, the ORF (open reading frame)region of the Pax6 variant genen was cloned successfully in RT-PCR. The PCR products were ligated into the T-ends of the PMD18-T vector following standard protocols. The constructs of Pax6 variant were digested from PMD18-T and then ligated into pET28-a vector. The recombinant expression plasmid was used to transform the strain of E.coli Rosseta (DE3).After identified the optimal condition for expression of the His-Pax6 variant, we figured out the optimal expression temperature is 20℃,the IPTG concentration is 0.5 mM. The protein were purified of large scale by the His affinity chromatography in the optimal condition. After analysised of the concentration and purification of the purified protein, mices were immunized with the protein to prepare its antiserum. ELISA shows that the antibody titers reached to 1: 64000.Purified the polyclonal antibody by Immunoaffinity Chromatography, And the sensitivity and specificity of the antibodies were evaluated by enzymelinked immunosorbent assay (ELISA) and Western blot analysis. ELISA shows that the antibody titers reached to 1:32000. Western Blot assay shows the purified antibody have higher specificity. Sequence alignments and secondary structure predictions of Pax6 variant and Pax6 wild type protein were performed using the 3D-PSSM sever. The fold recognition sever 3D-PSSM predicts a similar helical/sheet content for Pax6 variant and Pax6 wild type protein. Pax6 wild type protein comprises only 3% sheet and 25% helix, while the proportion in Pax6 variant protein is 5% sheet and 20% helix. Pax6 variant protein loss 2 helix motifs than Pax6 wild type protein.The truncated PST domain of Pax6 variant may lose their transactivation ability, resulted in the dominant-negative effects.Our research will lay the foundation for the mechanism of Pax6 variant gene and its encoded protein in the Bufo raddei Strauch development.