重组人睾丸精子结合蛋白对蛋白激酶A活性的影响

为探讨人睾丸精子结合蛋白(TSBP)在精子获能及顶体反应中可能发挥的作用,检测了TSBP重组蛋白在体外转染细胞系中对蛋白激酶A活性的影响。构建真核表达载体pcDNA3.1/myc-His(-)B/tsbp,稳定转染HEK293细胞后,蛋白免疫印迹检测到培养细胞中融合蛋白的表达。选择表达量高的细胞克隆,扩增并用金属亲和层析柱(IMAC)从细胞裂解液中纯化融合蛋白His6-TSBP。用放射自显影法检测HEK293细胞中蛋白激酶A(PKA)活性,发现重组质粒转染组PKA活性高于对照组,确定了TSBP对PKA活性的促进作用。

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.