ObjectiveTo construct prokaryotic expression plasmid pGEX-4T-1/SH3 and to examine the biological activity of the expressed SH3 protein. MethodsHCK SH3 gene was amplified by PCR and was cloned into the vector pGEX-4T-1 to construct prokaryotic expression plasmid pGEX-4T-1/SH3. The recombinant plasmid was identified by double enzyme digestion and sequencing, the positive plasmid was transformed into E. coli BL21 (DE3),and the expressed product was identified by SDS-PAGE electrophoresis and Western blotting analysis. HCK SH3 and HIV-1 Nef proteins were purified and their binding activity was detected by GST pull-down assay. ResultsThe recombinant plasmid pGEX-4T-1/SH3 was correctly constructed. SH3 protein was expressed and purified.GST pull-down assay showed that SH3 protein had a satisfactory binding activity to HIV-1 Nef protein.ConclusionWe have successfully expressed and purified GST-SH3 protein. Nef protein and SH3 proteins have a specific binding activity, which paves a way for screening drugs targeting Nef and SH3.