为研究牛源的化脓隐秘杆菌溶血素(PLO)生物学功能,本研究应用PCR方法扩增牛源化脓隐秘杆菌(A.pyogenes) PLO全基因序列,通过生物信息学方法分析其与猪源A.pyogenes的PLO蛋白差异,并构建了溶血功能区重组表达质粒pQE30-PLO585,在E.coli XLlBlue中用IPTG诱导表达.结果表明,扩增到PLO蛋白基因ORF为1 605 bp,编码535个氨基酸,与猪源PLO的核苷酸序列同源性为97.4％,氨基酸同源性为97.2％,在生物学活性功能区没有发生改变.表达的PLO蛋白溶血功能区重组蛋白能够被阳性血清识别,而且具有溶解绵羊红细胞的活性,产生β溶血现象.本研究获得了牛源A.pyogenes截短重组PLO蛋白,并证明PLO具有β溶血功能与较好的抗原性.%To express and identify the hemolytic activity of pyolysin (PLO) of Arcanobacterium pyogenes isolated from cattle, the complete gene of PLO was amplified by PCR, and the PLO gene from cattle and swine A. Pyogenes strains were analyzed by bioinformatics methods. The expression recombinant plasmid with truncated PLO585 was constructed, and expressed by IPTG induction in E. Coli XLlBlue. The results shown that the complete ORF of PLO gene was 1 605 bp encoded 585 amino acids. The homology of the nucleotide and the amino acids sequences of the PLO were 97.4% and 97.2% between bovine and swine A. Pyogenes strains, respectively, without change in biologic activity domains. The truncate PLO containing haemolysis domain was expressed in E. Coli which was able to react with positive serum and cursed (3 -hemolysis of sheep erythrocyte.
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