Purification, Characterization of Seminal Fibronectin Fragment in Human Seminal Plasma and Its Apllication for Legal and Clinical Medicine

从人精浆中分离得到一个36kDa蛋白，采用蓝-sepharose亲和层析、sephadex G-100凝胶过滤和Fast protein Liquid chromatography System进行纯化。该蛋白在12% sds -聚丙烯酰胺凝胶电泳中呈单条带。通过自动Edman降解对纯化蛋白进行测序。该蛋白的n端序列与人纤维连接蛋白细胞附着域1230 ~ 1237残基序列完全一致。说明36kDa蛋白为纤连蛋白片段。将纯化蛋白经完全弗氏佐剂乳化后免疫家兔制备抗精囊纤维连接蛋白片段血清。在人正常精浆（n-54）中，精纤维连接蛋白片段的浓度范围为316 ~ 2190 ug/ml（平均-996 ug/ml）。用抗精囊纤维连接蛋白片段血清，ELISA检测纯化血浆纤维连接蛋白的交叉反应。采用酶联免疫吸附法和免疫组化方法，对男性生殖器官精囊内的精纤维连接蛋白片段进行定位检测。

A 36kDa protein was isolated and purified by affinity chromatography utilizing blue-sepharose, gel filtration on sephadex G-100 and Fast Protein Liquid Chromatography System from human seminal plasma. The protein have a single band in 12% SDS-polyacrylamide gel electrophoresis. The purified protein sequenced by automated Edman degradation. The N-terminal sequence of the protein was found to corresponded exactly with the sequence of residues 1230 to 1237 at cell attachment domain of human fibronectin. The fact suggest the 36kDa protein was fibronectin fragment. The anti-seminal fibronectin fragment serum was prepared by immunizing rabbit the purified protein emulsified with complete Freund's adjuvant. In human normal seminal plasma (n-54), concentration range of the seminal fibronectin fragment was 316-2,190 ug/ml (mean-996 ug/ml). Cross-reaction was found the purified plasma fibronectin by ELISA using anti-seminal fibronectin fragment serum. By ELISA and immunohistochemical method, the localization of seminal fibronectin flagment in male genital organs was detected in seminal vesicle.