睾丸表达新基因HSD-28的克隆及其编码蛋白质的研究

Characterization of a Novel Human Testis-specific Gene——HSD-28 and Its Encoding Protein

目的研究从人初级精母细胞特异EST文库中克隆的新基因HSD-28及编码蛋白质在精子发生过程中的表达及可能发挥的功能。方法利用根据本实验室构建的人初级精母细胞特异EST文库,通过电子克隆方法,从网络数据库资源获取基因HSD-28。纯化原核表达系统表达的HSD-28蛋白,以此免疫新西兰白兔制备针对HSD-28的特异性多克隆抗体。采用免疫组化的方法研究HSD-28在人睾丸组织中的表达定位。最后,利用酵母双杂交系统筛选与HSD-28相互作用的蛋白质。结果克隆得到了的HSD-28基因,提交GenBank获得登陆号为AY251533,全长为641个碱基,其中开放阅读框为504碱基,编码一个由168个氨基酸构成的蛋白质,命名为HSD-28蛋白。将构建的pET-30a-HSD-28重组质粒转化到BL21(DE3)宿主菌,表达并纯化了HSD-28蛋白,大小约为28kDa。免疫组织化学结果显示,HSD-28蛋白特异的定位于人精原细胞和早期初级精母细胞。筛选得到与HSD-28相互作用的两个阳性克隆45#和106#。结论从人睾丸cDNA文库中成功克隆得到HSD-28基因,其编码蛋白质表达定位于人精原细胞和初级精母细胞中,可能参与精子发生过程中减数分裂的调节。

Objective To characterize a novel gene HSD - 28 screened from a primary spermatocyte - specific ESTs library and its encoding protein. Methods We used the electronic cloning method to acquire full length of HSD - 28,prepared a specific rabbit polyelonal antibodies against HSD - 28, detected the expressing pattern of HSD - 28 in human testis sections by immunohistochemistry, screened a human testes cDNA library using HSD -28 as a bait in a yeast two hybrid system. Results The sequence of HSD - 28 was obtained from a human primary spermatocyte -specific ESTs library,and submitted to Genebank with accession number AY251533. HSD -28,the encoding product of HSD -28,consisted of 168 amino acids. This protein was expressed in human spermatogonia and primary spermatocyte. Two proteins putatively interacting with HSD - 28 were achieved from a human testes cDNA library with the yeast two hybrid systems. Conclusion The HSD -28 encoding protein is expressed in human spermatogonia and primary spermatocyte,which might play roles in regulating the meiosis of mammalian spermatogenesis.