Cloning Of The Mouse Bicaudual-C-1 Gene,Preparation And Application Of Anti-Bicc1 Polyclonal Antibody

The Bicaudal-C(Bic-C) gene was first discovered in Drosophila melanogaster, it's normal function is required for correct targeting of the migrating anterior follicle cells and for anterior-posterior patterning of the Drosophila oocyte.Mouse Biccl gene is a homologe of Drosophila Bicaudal-C gene,whose mutation causes a phenotype sembles with human polycystic kidney disease,but the details of Biccl involved in cystogenesis are still elusive.Here we cloned the full-length cDNA sequence of Biccl gene by nest PCR and digest- ligate methods,then constructed a recombinant eukaryotic expression plasmid pCMV-Tag 4A-Biccl.Based on the bioinformatics analysis results of mouse Biccl,the DNA fragment encoding N-terminal 139 amino acids(61 E-199A) and C-terminal 148 amino acids(711Q-858D) of Bicc-1 was obtained by PCR and was cloned into a prokaryotic GST-fusion protein expression vector.Fusion protein GST-Bicc 1-N/P- His6 were expressed after IPTG induction,and then purified from the total proteins of E.coli Rosetta cells transformed by the recombinant plasmids pGex-HisC3-GST- Biccl-N/C.We prepared two polyclone antibodies against mouse Biccl protein with high specificity by immunizing rabbit with these two purified fusion proteins.The specificity of the affinity-purified antibodies was examined by Western blot.We stained cultured cells and mouse kidney paraffin sections with this antibody and found that Biccl is mainly localized at cytoplasm.These work will lay a foundation for further research on the biological function of mouse Biccl gene.