猕猴和小鼠分泌片的分离纯化及某些生化特性

ISOLATION, PURIFICATION AND SOME BIOCHEMICAL PROPERTIES OF SECRETORY COMPONENT FROM MONKEY AND MOUSE

为了深入了解分泌片和ＩｇＡ在粘膜免疫系统中的作用，自猕猴和小鼠胆汁中提取和纯化分泌片，ＳＤＳ－ＰＡＧＥ结果表明猕猴和小鼠游离分泌片的分子量均约为６０ｋＤａ，但在非解聚型聚丙烯酰胺梯度凝胶电泳时分子量分别为７４ｋＤａ和６２ｋＤａ。采用ＬＫＢ－８１００等电聚焦柱测定其ＰＩ范围，猕猴分泌片为４．３—５．９，小鼠分泌片为３．９—５．４。免疫双扩散证明，猕猴和小鼠胆汁中的分泌片均能与兔抗人分泌片免疫血清发生交叉反应。猕猴，小鼠胆汁中的分泌片及人乳汁中的分泌片与小鼠抗人精子ＩｇＡ型单抗（Ｋ０１４）进行体外组装后，以 Ｗｅｓｔｅｒｎ Ｂｌｏｔ显示三物种的分泌片均能与小鼠 ＩｇＡ单抗结合，其中小鼠分泌片的结合能力较强。

As an important glycoprotein in the mucosal immune system, secretory component (SC) can be associated with IgA and IgM ; SC is produced by epithelial cells of secretory glandular and mucosal tissues and by hepatocytes (of some species),and thereby mediates the transport of IgA and IgM into external secretions. After that,their resistance to protease cleavage increased. In order to know functions of antisperm monoclonal IgA assembled with or without SC in mucosal immune system,free SC from monkey and mouse bile were isolated and purified by chromatography on Sephadex G200 and DEAE-Cellulose 32 columns. The single band of purified SC from monkey and mouse was demonstrated both by SDS-PAGE and nondenatured gradient PAGE,and the variation of SC molecular weight was found in SDS-PAGE (60 kDa for both monkey and mouse SC)and in nondenatured gradient PAGE (74 kDa for monkey and 62 kDa for mouse). The PI range of monkey (4.3-5.9) and mouse (3.9-5.4)SC was measured by isoelectric focusing on LKB-8100 Ampholine column. SC from monkey and mouse may reacted with rabbit anti-human SC serum in double diffusion plate. The SC from human,mouse and monkey can be all combined in vitro with mouse antihuman sperm monoclonal IgA (K014),and the in vitro assembly was confirmed by Western blotting. The study on the biological function of assembled secretory IgA is underway.