单增李斯特菌溶血素(LLO)的原核表达及其生物学活性鉴定

利用生物软件设计单增李斯特菌溶血素蛋白的基因hly的引物,通过PCR扩增hly基因,并将其克隆至PET28a(+)原核表达载体,转化大肠杆菌BL21进行优化表达。用镍柱纯化表达产物LLO,通过免疫印记鉴定其免疫原性,并通过溶血实验鉴定其溶血活性。琼脂糖凝胶电泳结果表明PCR扩增出1 590 bp的片段,经测序鉴定其序列同源性可达99%。SDS-PAGE结果表明诱导表达的产物大小约为58 kD,其最优化的表达条件是28°C下用0.1 mmol/L IPTG诱导6 h。Western blotting结果表明重组表达的LLO具有免疫原性;溶血实验表明重组表达的LLO具有较强的溶血活性,其溶血效价可达1:1 024。这为制备针对单增李斯特菌的单克隆抗体及其检测方法的建立奠定了基础。

Prokaryotic expression and bioactivity identification of hemolysin from Listeria monocytogenes

Designing the primers of hly gene by biological software,amplifying the hly gene from Lis-teria monocytogenes by PCR and constructing the gene into prokaryotic expression vector PET28a(),then transforming the gene into E.coli BL21and expressing optimally;purifying the recombinant prod-uct LLO by nickel affinity chromatography,testing the immunogenicity by Western blotting and the hemolytic activity by hemolytic assay.It was demonstrated by agarose gel electrophoresis that the cloned hly gene was 1 590 bp in length and the protein sequence got about 99% homology with that published in GenBank.SDS-PAGE indicated that the molecular weight was about 58 kD and the opti-mal expression condition was induced for 6 h with 0.1 mmol/L IPTG at the temprature of 28 °C.The result of Western blotting showed the recombinant protein LLO had immunogenicity.The hemolytic assay showed the product had the hemolytic activity,with the hemolytic titre of 1:1 024.These results would provide basis for the further studies on the development of monoclonal antibody against Listeria monocytogenes and the establishment of the dection methods.