人γ-精浆蛋白的亲和纯化及其糖基化分析

Purification of Human γ-Seminoprotein by Affinity Chromatography and Glycosylation Analysis

目的:从人精液中提纯γ-精浆蛋白并对其生物学特性进行鉴定。方法:应用饱和硫酸铵法从小鼠腹水中纯化出抗γ-精浆蛋白的单克隆抗体E4B7,将其共价交联到CNBr-Sepharose4B上,制备成免疫亲和层析柱,用该层析柱亲和纯化经饱和硫酸铵粗提的精液标本。纯化产物分别用SDS-PAGE、Western-blot及ELISA进行生物学特性鉴定,最后经PNGaseF、Endo-H酶消化后进行糖基化分析。结果:SDS-PAGE电泳表明纯化蛋白的相对分子量约为44kDa,Western-blot及ELISA鉴定显示该蛋白可与抗γ-精浆蛋白的单抗E4B7特异性结合。对纯化蛋白无论是单用Endo-H酶消化,还是同时用Endo-H酶和PNGaseF酶共同消化,消化产物电泳后相对分子量均降低到34kDa左右,而单独用PNGaseF酶消化后相对分子量没有改变。Western-blot及ELISA鉴定显示糖苷酶处理后的纯化蛋白仍可与单抗E4B7特异性结合。结论:成功纯化出N-糖基化形式的γ-精浆蛋白,这为下一步筛选人源化抗体奠定了纯的抗原基础。

Objective： To purify and identify γ-seminoprotein from human seminal plasma and analyze the glycosylation form. Methods： he mAbE4B7 against humanγ-seminoprotein were firstly purified from mice ascites by saturated ammonium sulfate precipitation, and then coupled to CNBr-Sepharose4B as the Ligand for generation of immuno-affinity chromatography column. The sperm derived from health people were primly purified by saturated ammonium sulfate precipitation, and then employed to the immuno-affinity chromatography column. The purified production was treated respectively with peptide N-glycosidase F （PNGase F ） and Endo-β-N- acetylglucosaminidaseH （Endo-H） deglycosylated enzyme. The resultant productions were identified by SDS- PAGE, Western-blot and ELISA analysis respectively. Results： The purified protein with a band of molecular mass 44kDa were got in SDS-PAGE, and it bind specifically to mAbE4B7 in Western-blot and ELISA assay. The molecular mass of the purified protein was recovered to be 34kDa with the treatment of deglycosylated enzyme Endo-H alone, or both Endo-H and PNGase F. Whereas there is no molecular mass change by treating with PNGase F alone. The deglycosylated production also bind specifically to mAbE4B7 in Western-blot and ELISA assay. Conclusion： specificγ-seminoprotein with correct N-glycosylation are successfully isolated and purified. This purified seminoprotein provides a good antigen basis for humanization of mAbE4B7 by guide-selection technology.