基于裂解酶与ATP生物发光法特异定量检测炭疽杆菌方法的研究  

Quantitative Specific Detection of Bacillus anthracis Based on Bacteriophage Lysin and Luminescence Techniques

作　　者：张博[1,2] 康怀兴[2] 米志强[2] 安小平[2] 张飞雄[1] 童贻刚[2] 

机构地区：[1]首都师范大学生命科学学院,北京100048 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2014年第1期9-12,20,共5页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2009AA02Z111);国家科技重大专项(2011ZX09401-023;2011ZX10004-001);病原微生物生物安全国家重点实验室项目(SKLPBS1113)

摘　　要：目的:原核表达炭疽杆菌噬菌体γ裂解酶(PlyG)和萤光素酶(Luc),结合这2种酶建立特异定量检测炭疽杆菌的方法。方法:PCR扩增得到带有His标签的裂解酶基因和萤光素酶基因,构建重组表达载体pET22b-plyG和pET22b-luc,转化至大肠杆菌BL21(DE3)并诱导表达,过镍柱纯化得到目的蛋白;利用裂解酶裂解和ATP生物发光定量检测蜡样芽孢杆菌RSVF1,与平板计数法对比建立线性关系。结果:表达了炭疽杆菌噬菌体γ裂解酶和萤光素酶,并建立了特异定量检测蜡样芽孢杆菌RSVF1的方法,与平板计数方法具有显著的线性相关。结论:因炭疽杆菌与蜡样芽孢杆菌RSVF1均对PlyG具有较强的敏感性,故本研究所建立的将炭疽杆菌噬菌体γ裂解酶与萤光素-萤光素酶系统相结合的检测方法对现场或临床定性定量检测炭疽杆菌提供了理论支持,具有良好的应用前景。Express the γ phage lysin（PlyG） and luciferase（Luc） by prokaryotic expression system and make use of these two enzymes to establish a method of quantitative specific detection of Bacillus anthracis. Methods： The histidine-tagged plyG gene and the histidine-tagged luc gene, obtained by PCR amplification, were cloned into the Escherichia coli expression vector pET22b to construct the recombinant expression vectors pET22b plyG and pET22b-luc. The recombinant expression vectors were transformed into E.coli BL21（DE3） and were in duced to express. The target proteins were purified by Nickel-affinity chromatography. Luminescence techniques us ing the recombinant Luc and PlyG were utilized to detect the B.cereus strain RSVF1. We compared the lumines cence method and colony counting method to see if there is a linear relationship between them. Results： The PlyG and Luc were successfully expressed, a method of quantitative specific detection of B.cereus strain RSVF1 has been established, which shows a significant linear correlation with the colony counting method. Conclusion： Both B.anthracis and B.cereus strain RSVF1 are sensitive to PlyG, so the detection method by using PlyG and lu ciferin-luciferase luminescence techniques provides theory support for field test or clinical detection of B.anthracis and has a good application prospect.

关 键 词：炭疽杆 噬菌体裂解酶 萤光素酶 ATP生物发光法 定量检测 

分 类 号：Q78[生物学—分子生物学] Q502