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SITE-DIRECTED MUTAGENESIS, ISOLATION AND PURIFICATION OF LISTERIA MONOCYTOGENES LISTERIOLYSIN O AND ITS IMMUNOGENICITY

文献类型： 外文期刊

作者： Zhao, Kai 1 ; Sun, Xiaofei 3 ; Zhao, Xinghai 1 ; Qi, Baomin 3 ; Tang, Xueming 1 ;

作者机构： 1.Shanghai Acad Agr Sci, Key Lab Agr Genet & Breeding, Shanghai 201106, Peoples R China

2.Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai 201106, Peoples R China

3.Fujian Agr & Forestry Univ, Coll Anim Sci, Fuzhou 350002, Peoples R China

期刊名称：JOURNAL OF FOOD SAFETY （ 影响因子：1.953； 五年影响因子：1.946 ）

ISSN： 0149-6085

年卷期： 2011 年 31 卷 4 期

页码：

收录情况： SCI

摘要： Listeria monocytogenes is a food-borne pathogenic bacterial species that can cause diseases in humans and animals. A rapid and reliable method for detection of L. monocytogenes is crucial in disease control. Listeriolysin O (LLO), encoded by the hly gene, is the most important virulence factor of L. monocytogenes and a useful identifying factor. We studied the protein structure of LLO with the aim of developing an antibody to be used in an enzyme-linked immunosorbent assay (ELISA) for detection of L. monocytogenes. By in silico analysis, a Leu52Phe modification of LLO sequence was predicted to increase its immunogenicity. The corresponding modification in the hly gene sequence was achieved by site-directed mutagenesis. The wildtype and mutated hly genes were cloned and expressed in Escherichia coli; the expression levels were 10 and 12%, respectively. Highly purified protein preparations of LLO and its Leu52Phe variant (similar to 37 kDa) were obtained and their purity values were both greater than 90%. Both proteins were used independently to immunize rabbits. The average optical density (OD) value obtained with the anti-Leu52Phe variant antibody (P2) was higher by 0.463 OD units compared with the corresponding value for the anti-LLO antibody (P1) by ELISA, which indicated that the mutation had indeed resulted in enhanced immunogenicity of LLO.

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