SDH-SV2C-L4融合蛋白的表达及山梨糖脱氢酶活性检测

Expression of the Fusion Protein SDH-SV2C-L4 and Detection of its SDH Activity

目的:构建SDH-SV2C-L4融合蛋白表达载体,在大肠杆菌中表达具有山梨糖脱氢酶(SDH)活性的融合蛋白。方法:将C亚型突触囊泡蛋白2大突环L4(SV2C-L4)基因与SDH基因以GGGS柔性接头连接,在大肠杆菌DH5α中表达;用NBT染色和DCIP脱色的方法检测融合蛋白的SDH活性。结果:DNA测序及SDS-PAGE结果显示构建了融合蛋白表达载体,并表达了SDH-SV2C-L4融合蛋白,相对分子质量约80×103;DCIP脱色及NBT染色均检测到融合蛋白的SDH活性。结论:与SV2C-L4融合的SDH仍具有活性,为下一步SV2C-L4活性检测方法的建立及SDH与SV2C-L4的其他相关研究奠定了基础。

Objective：To construct a fusion expression vector for SDH-SV2C-L4 and express the fusion protein in E.coli with sorbose dehydrogenase（SDH） activity.Methods：The gene of SDH and synaptic vesicle protein 2C （SV2C）-L4 were linked with a GGGS linker and cloned into pET22b（＋）.The fusion vector was expressed in E.coli DH5α.SDH activity was detected by NBT staining and DCIP decolorizing.Results：DNA sequencing and SDS-PAGE analysis demonstrated that the fusion expression vector was successfully constructed,and the SDH-SV2C-L4 fusion protein was expressed.SDH activity was confirmed by both NBT staining and DCIP decolorizing.Conclusion：Recombinant SDH-SV2C-L4 fusion protein with SDH activity was obtained,which laid foundation for further study on SV2C-L4 and SDH.