山溪鲵皮肤β-microseminoprotein样蛋白的分离纯化及其cDNA克隆  

Purification and molecular cloning of a β-microseminoprotein-like protein from the Batrachuperus pachunii skin

作　　者：韩曜平[1] 苏月菊[2] 饶定齐[3] 张志伟[1] 夏德全[1] 吴婷婷[1] 

机构地区：[1]南京农业大学无锡渔业学院,江苏无锡214081 [2]金陵科技学院动物科学与技术学院,江苏南京210038 [3]中国科学院昆明动物研究所,云南昆明650223

出　　处：《南京农业大学学报》2007年第3期94-99,共6页Journal of Nanjing Agricultural University

基　　金：中国科学院2003年"西部之光"项目

摘　　要：从山溪鲵(Batrachuperus pachunii)皮肤匀浆液中经过Sephadex G-50凝胶过滤、AKTA(Resource Q阴离子柱和反向高压液相C4柱分离纯化得到相对分子质量为12 000的蛋白。利用其N端氨基酸序列设计引物,从山溪鲵皮肤的cDNA中克隆并筛选到该蛋白的cDNA序列。该cDNA序列的开放阅读框为339 bp,编码113个氨基酸残基组成的蛋白。在BLAST数据库搜寻比对分析表明,该蛋白的氨基酸序列与来自人类及其他哺乳动物β-microseminoprotein蛋白具有约40%的序列同源性。这也是首次在两栖类动物皮肤中确认β-microseminoprotein。初级结构分析表明,该蛋白属于亲水性蛋白;多重序列比较显示,其氨基酸序列中的10个半胱氨酸位点及15个其他氨基酸位点与高等脊椎动物β-microseminoprotein中同种氨基酸有相同的位点。由此推测该蛋白属于β-microseminoprotein家族。A protein with a relative molecular weight of 12 000 was purified from skin homogenate of Batrachuperus pachunii by gel filtration chromatography, AKTA^ОR Resource-Q ion exchange chromatography and liquid chromatography of reverse phase high performance. N-terminal amino acid sequence was determined by Edman degradation. A full-length gene had been cloned from the cDNA of Batrachuperus pachunii skin. A 339 bp open reading frame corresponding to a deduced protein of 113 amino acid was con- tained. The initial BLAST search at NCBI showed that the protein encoded by the cDNA had a β-microseminoprotein domin and the closest hit to human and other mammalian β-microseminoprotein with 40% of identity. It was the first time to identification the β-microseminoprotein in skin of amphibians to date. The preliminary structure analyzing indicated that this protein was a hydrophilic protein ; comparised of Batrachuperus pachunii β-microseminoprotein precursor-like protein sequence to other speices, 10 cystenies and other 15 aminooacids were conserved and located at the same positions as those in amniote β-microseminoprotein. It suggests that the purified protein might be structurally belong to the β-microseminoprotein family.

关 键 词：山溪鲵 β-microseminoprotein 分离纯化 CDNA序列克隆 皮肤匀浆液 

分 类 号：Q959.52[生物学—动物学] Q51