Specific Antibody Preparation And Structural Analysis Of Juxtanodin

AIM: To analyze the coding sequence and structural features of Juxtanodin (JN), and to prepare and purify the polyclonal antibody of JN, to identify the function of JN in cell morphology and myelination in central nervous system (CNS). METHODS: N-terminal 170 amino acids of JN was amplified by PCR and then sub-cloned into the prokaryotic expression vector pET-41a(+). In order to produce GST-tagged fusion protein in E.coli BL21(DE3), the GST-JN170 fusion protein was induced with IPTG, then the fusion protein was purified by the Bulk GST purification modules. After purification, GST-JN170 fusion protein was used to immunize six rabbits following traditional method and rapid method. The antiserum was purified by affinity chromatography. Western blot was used to identify the specificity of JN antiserum and purified antibody. Immunohistochemistry and cellular immunofluorescence were used to identify the localization of JN in cells and its effect on cell morphology. The coding sequence and structural features of JN was analyzed by bioinformatics methods, verify the protease activity of JN by SDS-PAGE. RESULTS: Prokaryotic expression vector pET41a(+)-JN170 and eukaryotic expression vector pcDNA3.1myc-His(-)-JN were successfully constructed and confirmed by sequencing. SDS-PAGE indicated that GST-JN170 fusion protein was expressed in soluble form. JN170 antiserum was generated by immunization of fusion protein and then purified by affinity purification. Western blot results showed that both rapid and traditional method could generate specific antiserum, the rapid method obtained higher specificity compared with the traditional one. Cellular immunofluorescence indicated that JN predominantly localized in the cytoplasm and processes of cells. Bioinformatics analysis indicated that the C-terminal of JN sequence contains an actin-binding domain which belongs to the conserved sequence of the ERM protein family, the N-terminal of JN was different between species. Reduced and unreduced electrophoresis verified that JN didn't exist in dimeric form but with other decorations. CONCLUSION: Prokaryotic expression vector and eukaryotic expression vector of JN were successfully constructed and soluble GST-JN170 fusion protein was obtained. Specific JN170 antiserum and purified antibody was generated through rapid and traditional method. The localization of JN in cytoplasm and processes of cells implied that JN might play a great role in cell motility and morphology. This study laid a foundation for further research of the function of JN in the migration of oligodendrocyte and myelination in CNS.