【摘要】 目的 :用目的基因随机表位库方法寻找葡激酶 (straphylokinaseSAK)的显性表位。方法 :①SAK免疫BALB C小鼠 ,免疫亲和层析纯化抗血清后 ,抗SAK抗体用生物素标记 ;②构建SAK随机表位肽库 ,随机挑取 12个独立克隆测序 ,分析库DNA片段的分布和碱基含量情况 ;③以多抗为靶蛋白用克隆原位杂交法筛选肽库 ;④构建SAK的缺失突变体mSAK ,Westernblot分析mSAK的免疫反应性。结果 :①筛选肽库得到一个由 19个氨基酸组成的免疫显性表位区 ,命名为A1区 ;②mSAK不能与抗SAK多抗反应。结论 :用简便、有效的方法筛选得到了SAK的一个表位A1区 ,初步确定A1区是SAK引起免疫反应的重要区域。更多还原

【Abstract】 Objective:To map dominant antigenic determinants on SAK by gene-targeted fragmemt library . Methods:①The PcAbs specificly against SAK were produced by immunning BALB/C mice. Pu rified the antisera through a SAK-Sepharose 4B affinity chromatography column. The purified PcAbs were biotinylated for next step. ② After constructing SAK r andom epitope library we sequenced 12 isolated clones randomly to ensure its int egrity ,capability and randomness.③The library was screening by situ-clone hy bridization.④Constructed mSAK ,the A1 region deleted mutant of SAK ,and used Western-blot assay to identified its immunoreactivity. Results:①Got a dominant epitope at amino acid 71-89,called A1 region; ②Western-bl ot assay suggested that mSAK, a mutant SAK without A1 region., didn’t combine to the anti-SAK PcAbs.Conclusion:A dominant epitope of SAK was mapped successfully with a simple,effective method . [更多还原