摘要
为研究炭疽毒素致死因子(lethalfactor,LF)致死机理,更有效地防治炭疽,对LF基因1550～2324bp之间的片段进行了随机突变实验.通过细胞毒性实验筛选LF突变体库,结果获得5株活性降低突变体.进一步对单点突变体蛋白进行亲和纯化,首次得到了K518E、L519C两种突变蛋白,对其进行纯蛋白活性检测和竞争抑制实验,发现这两种突变蛋白活性显著降低,表明518位赖氨酸和519位亮氨酸对LF功能起着非常重要的作用.并且发现,K518E对野生型LF具有一定的抑制作用,对LF作用机理的研究有一定帮助.
Anthrax toxin is one of the two virulence factors of Bacillus anthracis and it is a binary bacterium toxin composed of protective antigen （PA）, lethal factor （LF） and edema toxin （EF）. Among of them, LF is the enzyme moiety lead to the death of the cell. To investigate key function amino acid of LF and study the lethal mechanism of lethal factor, random mutagenesis was carried out. The mutation library of the 774 bp fragment of lethal factor from site 1 550 to 2 324 bp was constructed using DiversifyTM PCR Random Mutagenesis Kit （Clontech） with some modifiication. Transformants oflefmutation library were induced by 0.2 mmol/L IPTG at 28℃ for 4 h and then T7 phage were added into the culture, incubating for further 2-3 h to lyse the bacterium. The lysis was centrifuged at 12 000 r/min for 8 min and the supernatant which contained the target protein was collected. To determine the activity of the mutants, the cell cytotoxicity assay was carried out. RAW 264.7 microphage cells were seeded into 96-well plates, 24 h ahead of experiments. When assaying, the medium was removed and 100μl DMEM medium containing 1.0 mg/L of purified recombinant and 10 μl of the lysis supernatant was added into each well. Then the plates were incubated at 37℃ for 4 h. The cell viability was determined by using the alamarBlue^TM （AbD Serotec） dye according to the manufacturer＇s direction. The mutants whose activities had decreased significantly were sequenced and further purified by Ni-histidine tag affinity chromatography. The cell cytotoxicity and competition binding assay were demonstrated to analyse the character of the mutants. Five mutants which had low biological activity determined through the cell cytotoxicity assay were obtained. Furthermore, two mutant proteins with single-site mutation, K518E and L519C, were purified for the first time. The cytotoxicity assay showed that the two mutant proteins lost activity significantly, that means, Lys518 and Leu519 played an important role in the function of lethal
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2009年第10期1306-1312,共7页
Progress In Biochemistry and Biophysics
基金
教育部重大项目基金资助(306013)~~
关键词
炭疽
致死因子
随机突变
细胞毒性实验
anthrax
lethal factor
random mutation
cytotoxicity assay
