Construction of Wild Type and Mutant ATP6V1B2 Ggene Vector and Preliminary Study on Function of ATP6V1B2 Mutation
XIN Feng
YUAN Yong-yi
GUO Wei-wei
Han Dongyi
DAI Pu
Abstract：Objective To construct a eukaryotic expression vector containing the wildtype and mutant ATP6V1B2 gene and green fluorescent protein gene PEGFP for potential utility in functional study of ATP6V1B2 gene-related deafness. Meth-ods wildtype (pIRES2-EGFP-ATP6V1B2) and mutant (pIRES2-EGFP-ATP6V1B2-c.1516C>T) eukaryotic expression plas-mids were constructed and verified via gene recombination,restriction enzyme digestion,fixed-point mutation and gene se-quencing. After transfecting 293 cells via liposomes, ATP6V1B2 gene expression was observed, hydrolysis activity of V-ATPase and transfer activity of H+were detected in the transfected cells. Results The recombinant genes were confirmed to contain the wildtype and mutant ATP6V1B2 gene with correct sequence. Aftwr transfecting into the 293 cells for 24h, green fluorescent signals were visible in cytoplasm. Forty eight hours after transfection, in cells infected by eukaryotic expression plasmids, V-ATPase hydrolysis activity reduced and the pH of lysosome increased. Conclusion The eukaryotic expression vector containing wildtype and mutant ATP6V1B2 and EGFP genes has been successfully constructed which can express in 293 cells. Preliminary functional study has confirmed that the c.1516 C>T mutation results in decrease of the V-ATPase hy-drolysis activity and H+transfer activity.
Keywords：ATP6V1B2PlasmidExpression
Publication Date：2013-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 335-339 )