Green Fluorescent Protein Recombinant Nisin as a Probe for Detection of Gram-Positive Bacteria  

作　　者：Xiqian Tan Ye Han Huazhi Xiao Zhijiang Zhou 

机构地区：[1]School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

出　　处：《Transactions of Tianjin University》2017年第4期334-339,共6页天津大学学报（英文版）

基　　金：supported by the National Key Technology Research and Development Program of the Ministry of Science and Technology of China (No. 2015BAD16B01);TianjinKey Technology Research and Development Support Program (No. 13ZCDNC01900)

摘　　要：A great amount of foodborne pathogens were Gram-positive (G+) bacteria, a threat to public health. In this study, considering the binding ability of nisin towards G+ bacteria and the stable fluorescent ability of EGFP protein, a fluorescent nisin–EGFP protein probe was constructed by a gene engineering method. Nisin and EGFP were used as the receptor and fluorophore, respectively, to detect G+ bacteria. The nisin and egfp gene were amplified separately according to the sequence published in GenBank using unique primers. The two genes were cloned into a pET-28b(+) vector resulting in a pET-28b(+)–nisin–egfp vector. The vector was transferred into Escherichia coli (E. coli) BL21 (DE3) for expression. The expressed protein was extracted, purified by a Ni–NTA column, and then tested by the SDS-PAGE method to confirm its molecular weight. Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus), and Micrococcus luteus (M. luteus) were used as the representations of G+ bacteria. E. coli O157, representing the gram-negative (G−) bacteria, was used as a negative control. The binding specificity of the recombinant protein was performed on two types of bacteria and then detected through fluorescent microscopy. The results indicated that the nisin–EGFP probe could detect G+ bacteria at 108CFU/mL. © 2017, Tianjin University and Springer-Verlag Berlin Heidelberg.A great amount of foodborne pathogens were Gram-positive(G+) bacteria, a threat to public health. In this study, considering the binding ability of nisin towards G+ bacteria and the stable fluorescent ability of EGFP protein, a fluorescent nisin–EGFP protein probe was constructed by a gene engineering method. Nisin and EGFP were used as the receptor and fluorophore, respectively, to detect G+ bacteria. The nisin and egfp gene were amplified separately according to the sequence published in Gen Bank using unique primers. The two genes were cloned into a pET-28b(+) vector resulting in apET-28b(+)–nisin–egfp vector. The vector was transferred into Escherichia coli(E. coli) BL21(DE3) for expression. The expressed protein was extracted, purified by a Ni–NTA column, and then tested by the SDS-PAGE method to confirm its molecular weight. Listeria monocytogenes(L.monocytogenes), Staphylococcus aureus(S. aureus), and Micrococcus luteus(M. luteus) were used as the representations of G+ bacteria. E. coli O157, representing the gram-negative(G-) bacteria, was used as a negative control. The binding specificity of the recombinant protein was performed on two types of bacteria and then detected through fluorescent microscopy. The results indicated that the nisin–EGFP probe could detect G+ bacteria at 10~8CFU/mL.

关 键 词：ANTIBIOTICS Bins Cloning Escherichia coli Fluorescence Genes Health risks PATHOGENS Probes Proteins Recombinant proteins 

分 类 号：R446.5[医药卫生—诊断学]