Abstract:

Objective To construct mutant CD2-associated protein (CD2AP) fluorescence expression vector and transfect into podocytes, and investigate the distribution of F-actin in podocytes. Methods Eukaryotic expression plasmid pcDNA6-V5-CD2AP site-directed mutagenesis for 160G>A heterozygous mutation in exon 2 of CD2AP was conducted, and identification was performed by sequencing. Wild-type and mutant recombinant plasmid pEGFP-C1-CD2AP fluorescence expression vectors were constructed with wild-type and mutant plasmid and pEGFP-C1 fluorescence expression vectors, respectively, and mouse podocytes were transfected. The distributions of F-actin in podocytes at different cell cycles were observed by fluorescence microscopy. Results Sequencing analysis revealed that sitedirect mutagenesis of 160 G in exon 2 of pcDNA6-V5-CD2AP eukaryotic expression vector CD2AP to A was performed, while there was no changes in the other bases. In the interphase of cell division, F-actin in untransfected podocytes lined in parallel as fiber bundle. F-actin in podocytes transfected with wild-type recombinant plasmid displayed thick, short dot-like structures mainly around the nuclei, while F-actin in podocytes transfected with mutant recombinant plasmid expressed along the cytoplasm membrane. In the metaphase of cell division, F-actin filaments transfected with wild-type recombinant plasmid aggregated encircling podocytes, while those transfected with mutant recombinant plasmid only emerged as a number increase in cytoplasm with some regional aggregation. Conclusion Wild-type and mutant recombinant CD2AP fluorescence expression vectors are successfully constructed. CD2AP gene 160G>A heterozygous mutation may lead to the cytoskeletal changes of F-actin in the interphase and metaphase of cell division of podocytes.

Key words: CD2-associated protein, F-actin, expression vector, podocytes, mutation