Preparation Of Polyclonal Antibody And Expression Of Pod1 In Medaka(Oryzias Latipes)

Pod1 gene has played an important role in sexual differentiation,gonad development, organs formation and immunity of mammalian.However, the function of Pod1 gene in development was known little in non-mammalian vertebrates. High titer, purity and specificity antibody can be used as an important tool in studying gene and protein function.Nevertheless there have been no reports regarding the Pod1 gene polyclonal antibodies preparation and immune localization in nonmammalian vertebrates. We used medaka as a model animal, the main results are as follows.1. Podl gene sequence of medaka was translated into peptide sequence,then analyzed and choosed correspond peptide sequence to be synthesized by bioinformatics software. HPLC and MS results show that peptide sequence was synthesized correctly.2. The polyclonal antibodies were prepared by immunizing New Zealand rabbits with the purified peptide coupled to KLH. the titer, purity and specificity of the polyclonal antibodies were confirmed by SDS-PAGE, ELISA and Western Blot.3. The result of Western Blot bands gray analysis by bioinformatics software shows that POD1 protein was expressed the most in liver and the least in intestine of adult medaka.4. POD1 protein expression was determined by immunohistochemistry and immunofluorescence, the results indicate that POD1 protein are expressed in hepatic plate, hepatic sinusoid and around central vein in liver; mucosal epithelium and intestinal wall in intestine; myocyte nucleus in muscle; membrane and cytoplasm especially around yolk nucleus in stage IV oocyte; granulosa cell and thecal cell especiallyaround yolk globule in stage VII oocyte; granulosa cell and yolk mass in stage IX oocyte; sertoli cell and epithelial cell of efferent duct and membrane in testis.The polyclonal antibodies were prepared successfully for the first time,these studies demonstrated that Pod1 gene was very important in immune defence and organs such as gonad development in medaka,which laid the foundation to further study of the molecular mechanism of Podl gene during tissue development and immune defence.