特异性表达于附睾的核糖核酸梅酶A超家族成员人类核糖核酸酶9是一种新的精子结合蛋白

摘要

为研究人类核糖核酸酶99(Rnase 9)的功能,我们构建了哺乳动物融合表达载体pcDNA-hRNase9,通过HEK293T细胞制备了人类核糖核酸酶9-His融合蛋白,并测定了该蛋白的N-端氨基酸序列.根据确定的成熟蛋白,通过E.coli制备了重组Rnase 9蛋白,检测了其核糖核酸酶活性和抗菌活性,采用间接免疫荧光技术分析了人类核糖核酸酶9蛋白在成年人体组织的表达以及在射出精子、体外获能精子表面的定位.结果显示Rnase 9蛋白对面包酵母tRNA缺乏核糖核酸酶活性,而对E coli XLIBlue具有杀菌作用,并表现出剂量依赖和时间依赖的方式.间接免疫荧光法证实Rnase 9蛋白在成体附睾组织大量表达,而在检测的其他成体组织没有表达,在射出精子和体外获能精子的表面则集中分布于整个头部和颈部.这些结果表明Rnase 9蛋白可能在男性生殖道的宿主防御中发挥重要作用.%To explore the functions of human ribonuclease 9 (RNase 9), we constructed a mammalian fusion expression vec-tor pcDNA-hRNase9, prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences. According to the determined mature protein, recombinant human RNase 9 was prepared in E. coll. Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detect-ed, and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated sperma-tozoa were analyzed via indirect immunofluorescence assay. The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA, but exhibited antibacterial activity, in a con-centration/time dependent manner, against E. coll. Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis, but not present in other tissues examined, and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermato-zoa. These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.