精子发生相关基因片段CG14的克隆及其蛋白的表达

Cloning and Expression of a Novel Gene Fragment CG 14 Related to Spermatogenesis in E. coli

为研究与精子发生相关的基因并探讨其功能 ,用差异显示法发现了 1个与精子发生相关的基因片段CG14 .将该基因片段克隆到表达载体pGEX 3X上 ,在大肠杆菌中表达了融合蛋白 .通过谷胱甘肽 Sepharose 4B亲和柱纯化该融合蛋白 .经Xa因子酶切后Western印迹方法证明 ,靶蛋白分子量约为 8kD ,与预期分子量相符 .用融合蛋白免疫家兔获得抗血清 .免疫印迹实验表明 ,血清中含有CG14蛋白的特异性抗体 。

Based on previous work, a novel gene fragment CG 14 related to spermatogenesis was cloned into the prokaryotic expression vector pGEX 3X. After restriction analysis,PCR identification and DNA sequencing, the positive clone was induced by IPTG. The expected protein was expressed in fusion form with a high yield. The expression product was purified by glutathione Sepharose 4B affinity chromatography and digested by factor Xa. Western blotting showed that the approximate molecular weight of the final product was 8 kD. Rabbits were immunized with the fusion protein, the specific antibody against the target protein was raised.