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生物信息分析化脓性链球菌溶血素O(streptolysin O,Slo)蛋白结构表明,Slo蛋白除含有由461氨基酸残基组成的溶血活性结构域Thiol_cytolysin外,在N端还有一跨膜结构域。利用pET101-GENE蛋白表达系统,成功构建出表达具有Slo活性重组蛋白的重组子,采用镍柱亲和层析分离技术,纯化目的蛋白;纯化蛋白SDS-PAGE检测分析表明,重组蛋白与预测的溶血活性结构域的分子量相一致;溶血实验显示,纯化重组蛋白具有溶血活性。以纯化的重组蛋白为免疫原,对大鼠进行4次免疫,所获得免疫血清经Elisa检测,抗Slo血清效价达到1∶12 800;Western blot检测猪链球菌、马链球菌和化脓性链球菌中的链球菌溶血素结果显示,抗Slo多克隆抗体仅能与化脓性链球菌溶血素O发生反应,表明研究制备的化脓性链球菌溶血素O活性结构重组蛋白抗原具有较好的特异性,所制备的抗原Slo可用于进一步开发抗链球菌溶血素O(ASO)试剂盒。
Bioinformatics analysis Streptolysin O (Slo) protein structure of Streptococcus pyogenes showed that in addition to Thiol_cytolysin, a hemolytic domain consisting of 461 amino acid residues, the Slo protein has a transmembrane domain at the N-terminus. The pET101-GENE protein expression system was used to construct a recombinant expressing the recombinant protein with the activity of Slo. Nickel column affinity chromatography was used to purify the recombinant protein. SDS-PAGE analysis showed that the recombinant protein and the predicted hemolysis The molecular weight of the active domain is consistent; hemolysis experiments show that the purified recombinant protein has hemolytic activity. The purified recombinant protein was used as the immunogen to immunize rats for 4 times. The obtained immune serum was detected by Elisa and the titer of anti-Slo serum reached 1:12 800. Western blot was used to detect Streptococcus suis, Streptococcus equi and purulent chain Streptococcus hemolysin in cocci showed that anti-Slo polyclonal antibody only reacted with S. pyogenes O, indicating that the constructed recombinant S. tuberculosis hemolysin O active recombinant protein antigen has good specificity , The prepared antigen Slo can be used to further develop the anti-streptolysin O (ASO) kit.