李斯特菌溶血素基因的原核表达及其生物学特性

李斯特菌溶血素(LLO)是产单核细胞李斯特菌的主要毒力因子，利用PCR技术从血清型4b的产单核细胞李斯特菌菌株中扩增出编码LLO的hly基因，经克隆筛选和测序鉴定后，构建成该基因的原核表达质粒pGEX6P1hly，SDSPAGE结果表明：LLO与谷胱甘肽在大肠杆菌中已融合表达，融合蛋白的分子量为82kD；溶血实验证明融合蛋白具有较强的裂解真核细胞膜的作用，表明表达产物LLO具有生物活性，其溶血效价达2.26×101.4 HU/mg，这为进一步研究其致病与免疫机理、单抗研制和疫苗设计提供了条件。

Prokaryotic Expression and Bioactivity of The Listeria monocytogenes Listeriolysin O

To express the Listeria monocytogenes hly gene in Escherichia coli and study its primary biological characteristics, hly gene without signal peptide sequence was amplified from Listeria monocytogenes serotype 4b by PCR and inserted into T-easy vector. Sequencing showed this hly gene was 1518 bp and nucleotide homology was more than 99.8% compared with three Listeria monocytogenes hly genes in GenBank. The cloned hly gene was then inserted into prokaryotic expression vector pGEX-6P-1 and transformed into E. coli BL21. The predicted fusion protein was detected by SDS-PAGE after IPTG inducion, which had molecular weight approximately 82 kD. Hemolytic experiment demonstrated the expressed fusion protein can lyse human red cell and its hemolytic titer attained 2.26 x 10(4) HU/mg. Conclusively, the Listeria monocytogenes hly gene was successfully cloned and expressed in E. coli. The successful expression of LLO in E. coli BL21 constituted a solid foundation for further researches such as pathogenesis and immune mechanism, MAb and vaccine development.