青海弧菌lux基因在大肠杆菌中的异源表达

Heterologous expression of lux gene from Vibrio qinghaiensis in E.coli

研究淡水发光菌青海弧菌Vibrio qinghaiensis Q67的lux CDABE发光基因在大肠杆菌中的表达,并测定其对相关因素的耐受性,为其应用条件的筛选提供有利依据.对构建好的重组菌进行酶切验证筛选出发光重组菌,将其在含氯霉素的液体LB培养基中以异丙基硫代-β-D-半乳糖苷(IPTG)为诱导剂,对发光特性的表达条件进行研究,考察重组菌生长与发光的对应关系,癸醛、p H、温度和IPTG对发光的影响,以及重组菌在室温下的稳定性测定.结果显示,重组菌发光强度随菌体生长先升后降,在对数生长后期发光强度达到最大;当癸醛浓度(0.1-3.0μL/m L)、p H(3.0-11.0)、温度(25-43℃)和IPTG(0.05-0.35 mg/m L)从小到大变化时,重组菌的光强均呈现先增后减趋势;各影响因素分别为癸醛浓度1.0μL/m L,p H 6.0,温度37℃,IPTG浓度0.10 mg/m L时,重组菌发光最强;发光在10min内达到最大值后迅速下降,后来平缓波动趋于稳定,3 h内光强仍在测量范围内.本研究表明,青海弧菌lux CDABE基因在大肠杆菌中能很好表达并维持发光,重组菌对环境的耐受性为lux基因作为发光标记基因提供了应用范围参考.

This research aimed to investigate the recombinant Escherichia coli TOP10/pHSG396-luxCDABE expressing lux genes from a freshwater luminescent bacterium Vibrio qinghaiensis Q67, and to study the luminescence expression conditions of the transformants. We first screened the luminous recombinant strains with restriction digestion. Then we analyzed the corresponding relationship between the recombinant growth and luminescence curve, the effects of decanal, pH, temperature and IPTG on luminescence, and the stability of recombinant bacteria. The results showed that under the conditions of 0.1-3.0 μL/mL decanal, pH 3.0-11.0, 25-43 ℃ and IPTG 0.05-0.35 mg/mL, the luminous intensity first rose then descended. The maximum intensity occurred in the logarithmic anaphase, at the level of 1.0 μL/mL decanal, pH6.0, 37 ℃ and 0.1 mg/mL IPTG. At room temperature, the luminous intensity peaked at the tenth minute, then decreased sharply, with a flat fluctuation at approximately the third hour. The results indicated that luxCDABE genes from Vibrio qinghaiensis Q67 can express well in E. coli, and are suitable for bioluminescence reporter use.