青海弧菌Q67荧光素酶基因luxAB与luxCDABE在大肠杆菌中表达与发光特性比较

Comparison of the expression and luminescence of luxAB and luxCDABE genes from Vibrio qinghaiensis sp.—Q67 in Escherichia coli

中文关键词:  青海弧菌  荧光素酶基因  重组菌  发光特性

英文关键词:Vibrio qinghaiensis  luciferase genes  recombinant bacterias  performance

中文摘要:

      目的 对比淡水发光细菌——青海弧菌Q67荧光素酶基因luxAB和luxCDABE在新的表达体系中的表达, 为构建特异性检测毒性的重组菌提供基础。方法 设计引物扩增得到青海弧菌Q67荧光素酶基因luxAB和luxCDABE, 将其分别与表达载体pET-22b连接, 转化大肠杆菌DH5α测序, 阳性质粒转化大肠杆菌BL21(DE3)进行表达。检测两种重组菌的发光特性及重金属离子对其毒性影响。结果 通过测定重组菌的相对发光强度(RLU)和OD600值可知: luxAB和luxCDABE重组菌在癸醛和IPTG浓度均为1 μL/mL培养基里培养第1小时后达到最大RLU, 值分别为750618.5和140901.5。检测重金属离子Cd2+、Hg2+、Cr6+对重组菌的毒性, EC50(半有效浓度)值分别为: luxAB重组菌: 5.160、5.129、7.097 mg/L; luxCDABE重组菌: 6.137、7.643、7.736 mg/L。结论 在相同培养条件下, luxAB重组菌比luxCDABE重组菌获得了更大的RLU, 说明luxAB表达效果更好。重金属对重组菌的毒性影响检测, luxAB重组菌得到了更低的EC50值, 进一步论证了这个观点。

英文摘要:

      Objective To compare the expression of luxAB and luxCDABE genes from “freshwater luminescent bacteria”Vibrio qinghaiensis sp.—Q67 in Escherichia coli, and to make preparations for the construction of recombinant bacteria which could especially detect heavy metals. Methods Primers were designed in order to obtain the homologous fragments of luxAB and luxCDABE, then got the product through polymerase chain reaction (PCR). As the next process, the product was cloned into vector pET-22b and then transferred into Escherichia coli DH5α for genetic sequencing. The positive clones were propagated and the plasmids extracted were transferred into Escherichia coli BL21 (DE3). Performance of bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Vibrio qinghaiensis was detected. Results After about 1 h culture with 1 μL/mL decanal and 1 μL/mL IPTG, the luxAB and luxCDABE recombinant bacteria got the highest RLU: 750618.5 and 140901.5 respectively. EC50 of luxAB and luxCDABE recombinant bacteria of Cd2+, Hg2+, and Cr6+are 5.160,5.129,7.097 mg/L and 6.137, 7.643,7.736 mg/L respectively. Conclusion The expression of luxAB is more effective than luxCDABE in E. Coli. LuxAB recombinant bacteria have lower EC50 values, and the luxAB recombinant bacterias are more sensitive towards heavy metals.