单核细胞增多性李斯特菌LMO1847基因在不同应激条件下的表达

根据GenBank登陆的单增李斯特菌标准株EGD序列设计引物,PCR扩增LM01847基因片段后插入表达载体pET-30 a,构建重组原核表达载体,并在E. coliBL21中成功表达。纯化目的蛋白制备多抗,ELISA结果显示:该蛋白多抗与单增李斯特菌全菌、全菌多抗与该蛋白均可发生反应。证明该蛋白在天然状态下存在于单增李斯特菌表面,且具有良好的免疫反应性。该蛋白在45℃、高盐、酸性环境中表达水平基本稳定,但在低pH值条件下反应活性消失;高盐或低温条件下,表达量随盐浓度的升高或温度的下降而降低。除4℃以外,该蛋白均能保持良好的免疫反应活性。本研究为建立针对该蛋白的李斯特菌免疫微球凝集、免疫磁珠分离等检测方法奠定基础。

Expression of outer membrane protein LMO1847 of Listeria monocytogenes under stress conditions

A pair of primers was designed according to the sequence of LMO1847 of Listeria monocytogenes strain EGD from GenBank.The gene fragment of LMO1847 without signal peptide sequence was obtained by PCR from genomic DNA of L.monocytogenes,and cloned into prokaryotic expression plasmid pET-30a.The recombinant plasmid was transferred into E.coli BL21 and expressed successfully.The purified recombinant protein was used for polyclonal antibody production in rabbits.ELISA analysis showed that the polyclonal antibody of LMO1847 was reactive to whole listeial cells and the polyclonal antibody against the whole cells of L.monocytogenes was reactive to the recombinant LMO1847 protein.Dot-ELISA and the whole bacteria coated ELISA showed that LMO1847 existed on listerial cell surface with good immune reactivity.The expression level of LMO1847 remained stable under short period(<3 h)of elevated temperature at 45 ℃,5.5% NaCl or acidic(pH 5.5)stress conditions,but the reaction disappeared under acidic stress at pH 3.0.Expression level of LMO1847 by L.monocytogenes in these conditions was decreased depending on the concentration of salt and temperature variation,and the reaction ability wais steady except grown in 4 ℃.