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[The effect of recombinant TSBP on protein kinase A activity].

To investigate the function of testis sperm binding protein (TSBP) in sperm capacitation and acrosome reaction, the effect of the recombinant TSBP on the activity of protein kinase A was detected in the transfected cell line. With the use of prokaryotic expressing plasmid pGEX-5X-1/tsbp as template, the novel gene tsbp was amplified by PCR and a eukaryotic expressing vector pcDNA3.1/myc-His(-)B/tsbp was constructed. DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3.1/myc-His(-)B/tsbp had been constructed successfully. After the recombinant plamid being transfected into HEK293 cells, Western blot verified the expression of tsbp. Fusion protein His6-TSBP was purified from the cell lysate by immobilized metal-ion affinity chromatography (IMAC). Radioautograph revealed a higher PKA activity in the transfected HEK293 cells than in the control group, which indicates that TSBP can increase the activity of PKA.