【摘要】 研究大鼠L PGDS在毕氏酵母中的表达 ,探讨L PGDS在大鼠精子中的分布。以大鼠睾丸mRNA为模板 ,经逆转录获得L PGDS全长和成熟基因片段。将该基因片段克隆到pPIC9载体上 ,将重组表达质粒转化巴斯德毕赤酵母 (Pichiapastoris)GS115 ,筛选mut+ 表型 ,经甲醇诱导实现L PGDS的分泌表达。用SDS PAGE分析L PGDS的分子量 ,用PAS反应鉴定糖链 ,用免疫组织化学法研究L PGDS的分布 ,用Westernblot分析重组L PGDS与精浆中L PGDS。筛选出 4株表达水平较高的酵母工程菌株 ,SDS PAGE分析表明 ,产物分子量约为 2 7kD ;L PGDS主要分布在精子的头部以及尾部的上段更多还原

【Abstract】 To investigate the expression of rat L-PGDS by Pichia pastoris and to study the distribution of L-PGDS on sperm. The cDNA of rat Lipocalin-type prostaglandin D synthase was cloned by RT-PCR using mRNA of rat testis as template. The L-PGDS cDNA was inserted into plasmid pPIC9 and transformed into Pichia pastoris GS115 cells. Using SDS-PAGE to analysis the molecular weight of L-PGDS, immunohistochemical method was performed to study the distribution of L-PGDS on sperm, and Western blot method was performed to study the specific of L-PGDS. The molecular weight of recombinant L-PGDS is 27kD. L-PGDS is mainly distributed on sperm.更多还原