RSD-7的克隆及其在睾丸组织中的定位

Cloning and Characterization of a Novel Rat Gene RSD-7 Differentially Expressed in Testis

目的分离、克隆精子发生相关基因,深入了解精子发生的分子机制。方法采用本组克隆的大鼠睾丸EST筛选cDNA文库、Northernblot、原核表达和纯化、抗体制备和免疫组化等技术。结果(1)获得1个新的全长cDNA序列,命名为RSD-7。全长1238bp,编码232个氨基酸,GenBank接收号为AF315467。序列分析显示,RSD-7基因编码蛋白质含有1个Ubiquitin-like结构域。(2)Northernblot结果显示,在8种成鼠组织中,RSD-7基因仅在睾丸组织中有明显表达。不同发育天龄的大鼠睾丸组织Northernblot结果显示,出生后30d的大鼠RSD-7基因开始表达,一直到120d仍有表达。(3)RSD-7cDNA在大肠杆菌中获得高效表达,并纯化了GST-RSD-7融合蛋白,以此为抗原制备了高效价的抗RSD-7蛋白的多克隆抗体。(4)通过免疫组织化学方法,RSD-7蛋白定位于Sertoli细胞胞浆和质膜上。结论初步分析了RSD-7基因的表达特征并制备了抗RSD-7多克隆抗体,为进一步研究RSD-7蛋白在精子发生过程中的功能提供了条件。

Objective To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis.Methods Screening of the cDNA library,Northern blot,expression and purification in E.coli with GST expression system,immunocytochemical staining of testis sections were used.Results(1)A cDNA fragment designated as RSD-7was isolated from rat testis cDNA library.It was1238bp in length,coding a protein of232amino acids with the GenBank accession number AF315467.The encoding protein of RSD-7cDNA had a Ubiquitin-like domain.(2)Northern blot indicated that RSD-7was uniquely expressed in rat testis,and in the testis RSD-7emerged on the30 th postnatal day and expressed until120 th postnatal day.(3)Expression and purification of RSD-7protein in E.coli with GST expression system and were used to obtain anti-RSD-7antibody.(4)Immunolocalization of RSD-7in rat testis revealed that it is expressed only in Sertoli cells.Conclusions Transcription pattern of RSD-7and localization of RSD-7protein in testis have been made,which established the base for the functional study of RSD-7.