Metridia荧光素酶检测条件的优化

目的：探讨培养基pH、血清对Metridia荧光素酶（MLuc）检测的影响并尝试找到一种优化的检测条件。方法：构建表达MLuc的逆转录病毒载体，瞬时转染人宫颈癌细胞系HeLa，制备收获MLuc；利用化学发光仪检测不同条件下的培养基对MLuc发光性能的影响，并尝试通过不同缓冲液与试剂的组合代替培养基来获得最佳条件。结果：培养基pH变化导致MLuc发光值产生较大波动，血清的存在会导致检测背景；添加0.02%（V/V）NP-40的磷酸盐缓冲液（PBS）（pH=7.4）作为MLuc检测的缓冲体系代替培养基，其发光值大小相当，检测背景值由原来的600左右降低到20左右。结论：以添加0.02% NP-40的PBS作为MLuc检测的缓冲体系可消除培养基pH、血清的影响，使检测结果更加准确和灵敏。

A modified assay system for Metridia luciferase

OBJECTIVE: To investigate whether the pH and serum of cell culture medium would affect the Metridia luciferase (MLuc) assay or not,and to explore a modified assay method for MLuc. METHODS: A recombinant retrovirus vector which expressed MLuc was constructed and then transiently transfected into human cervical cancer cell line HeLa to produce MLuc. Then,the effects of cell culture medium (e.g. buffers combined with reagents) on bioluminescence of MLuc were assayed. RESULTS: Various pH levels of cell culture medium caused great fluctuation of the bioluminescence intensity of MLuc. In addition,serum in the cell culture medium would alter the background of MLuc. When the cell culture medium was replaced by phosphate buffered saline (PBS) supplemented with 0.02% (V/V) NP-40 (pH=7.4),the bioluminescence intensity between them was comparable. However,the background level of MLuc was decreased from 600 to 20. CONCLUSION: Using PBS supplemented with 0.02% NP-40 as an assay buffer for MLuc,the side effects from pH and serum of cell culture medium during MLuc assay would be reduce and application of MLuc in research can be improved.