人源鞘氨醇-1-磷酸裂解酶载体构建及表达分析

Cloning and Expression of Human Sphingosine-1-phosphate Lyase

目的:为深入研究作为疾病靶点的人源鞘氨醇-1-磷酸裂解酶(hSPL)的功能及机制,构建原核表达载体以表达得到具有活性的hSPL用于后续的研究。方法:首先将hSPL基因的全长(SPL-C)和跨膜区外的截短片段(SPL-T)构建到pET28b表达载体。用Western Blot和酶活检测比较大肠杆菌JM109、DH5α、Rosetta(DE3)、BL21(DE3)4种感受态细胞的表达情况。结果:成功构建hSPL原核表达载体,SPL-T在大肠杆菌Rosetta(DE3)和BL21(DE3)高表达,并且具有很强的活力。结论:得到在大肠杆菌Rosetta(DE3)和BL21(DE3)表达具有活力的hSPL-T裂解液上清,为下一步深入研究hSPL的酶学性质提供了很好的研究基础。

Objective： For further research of human sphingosine - 1 - phosphate lyase （hSPL） as a target for many kinds of disease, it is necessary to obtain active hSPL to study its function. Method： Construction of full -length and truncated hSPL gene to pET28b vector, analysis of the protein expressed in E. coli JM109, DH5α, Rosetta （DE3） and BL21 （DE3） competent cells by Western Blot and fluorescence activity assay. Result： Construction of the hSPL expression vector is successful. Expression level of hSPL is higher in E. coli Rosetta （DE3） and BL21 （DE3） than in E. coli JM109 and DH5α. The truncated protein shows high activity. Conclusion： We obtained the active superuatant of hSPL - T lysate which provides a basis for functional studies of hSPL.