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

采用蓝色琼脂糖亲和层析、Sephadex G-100凝胶过滤和Fast Protein高效液相层析系统，从人精浆中分离纯化出一种36 kDa的蛋白质。该蛋白在12%的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法中为单一条带。用自动Edman降解法对纯化的蛋白质进行测序。该蛋白的N-末端序列与人纤维连接蛋白细胞粘附区的1230-1237位氨基酸序列完全一致。提示该36 kDa蛋白为纤维连接蛋白片段。纯化蛋白经完全弗氏佐剂乳化后免疫兔，制备抗精液纤维连接蛋白片段血清。在人正常精浆(n-54)中，精液纤维连接蛋白片段的浓度范围为316-2,190 ug/ml(平均-996ug/ml)。纯化后的血浆纤维结合蛋白与抗精浆纤维结合蛋白片段血清发生交叉反应。采用酶联免疫吸附试验和免疫组织化学方法，检测精液纤维连接蛋白在男性生殖器中的定位。

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.