Initial Study Of The Function NBEAL1 Gene And Protein

Objective:To clone human NBEAL1 gene fragment, express and purify matched peptides, identify purified GST-tagged NBEAL1 peptides, prepare matched monoclonal antibody, and detect the expresstion levels of NBEAL1 in different grades of glioma.Methods: The human NBEAL1 gene fragments were obtained from the total RNAs of human glioma cell U251 by RT-PCR, cloned into the pGEX-KG expression vector, and transferred into E. coli BL21 (DE3). The expression of NBEAL1 peptides was induced by IPTG, and purified by GST affinity chromatographic column. The bioactivity of purified peptides was identified by Western Blotting. Matched monoclonal antibodies were prepared by fusing SP2/0 cells with spleen cells of BALB/c mice immunized with purified NBEAL1 peptides.Results: The NBEAL1 gene fragment was obtained by RT-PCR, the NBEAL1 peptide was expressed in inclusion bodies with a yield of more than 30% of total bacterial proteins, and the purity of purified NBEAL1 peptide was more than 95%. Western Blotting confirmed the purified proteins own bioactivity. Its matched monoclonal antibiodies were prepared, immunohistochemical analysis showed NBEAL1 expression was low in normal brain tissue but increased significantly in gliomas with low grades. However, its expression decreased in the counterparts with higher grades.Conclusion: The NBEAL1 gene fragment was successfully cloned, expressed, purified and confirmed to own bioactivity. Its monoclonal antibody was successfully prepared. NBEAL1 participates in the development and progression of gliomas. Our research lays foundation for clarifying its function in the initiation and progression of human glioma.