利用重叠延伸PCR构建红色荧光蛋白布鲁氏菌表达载体

doi:10.16431/j.cnki.1671-7236.2018.09.006

Abstract

Abstract:

To construct red fluorescent protein expression vector of Brucella host cells and individuals,RBS-Red and BDNA (Brucella DNA) fragments were obtained by PCR,and the RBS-Red-BDNA overlapping fragments were obtained by overlapping extension PCR method in this experiment.The overlapped fragments were inserted into pLB vector,the recombinant pLB vector and pMC-221 plasmid were digested by Sma Ⅰ and Sac Ⅱ double enzyme,the ligation fragments were transformed into E.coli DH5α,the extract plasmid was electroporated into Brucella 16M competent cell,16M-pMC-Red strain was coated with Brucella culture medium containing chloramphenicol resistance,and observed the strain color under inverted fluorescence microscope.The 16M-pMC-Red strain was used to infect the mouse macrophages and make the cell crawling slices,observation of mouse macrophages under laser confocal fluorescence microscopy and identification of red fluorescent expression.The results showed that the double promoter expression vector of red fluorescent protein in Brucella was successfully transformed by overlapping extension PCR method,the transformed plasmid was electroporated into Brucella strain 16M,and the strain could stably express the red fluorescent protein.The 16M-pMC-Red strain in mouse macrophages could stably express the red fluorescent protein.The red light-producing plasmid pMC-Red could be used together with the green light-emitting plasmid pMC-221,which laid a foundation for the research of different strains of Brucella,and also provided a new strategy using fluorescence detection as an indicator for the detection of brucellosis.

Key words: Brucella; red fluorescent protein; plasmid transformation; infection

CLC Number:

S852.61⁺4

LUO Cheng, QI Jiangjiao, WANG Yuanyuan, GAO Jianfeng. Construction of Red Fluorescent Protein Expression Vector of Brucella Using Overlapping Extension PCR Method[J]. , 2018, 45(9): 2394-2400.

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Construction of Red Fluorescent Protein Expression Vector of Brucella Using Overlapping Extension PCR Method

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